ES2972793T3 - Cells designed to induce tolerance - Google Patents

Abstract

The present invention relates to cells that are engineered to increase the cellular level of cellular FLICE [Fas-associated death domain-like IL-1 β-converting enzyme (FADD)]-inhibitory protein (CFLAR). The engineered cells have the ability to induce tolerance. Improved tolerogenicity is useful for prolonging the survival of a foreign transplant and for the treatment of autoimmune diseases. (Automatic translation with Google Translate, no legal value)

Description

DESCRIPTION

Cells designed to induce tolerance

Technical field

The current teaching relates to cells that are engineered to increase the cellular level of cellular FLICE [Fas-associated death domain (FADD)-like IL-1 p converting enzyme] inhibitory protein (CFLAR). The engineered cells have the ability to induce tolerance. Improved tolerogenicity is useful for prolonging foreign transplant survival and for treating autoimmune diseases.

The present invention relates to populations of cells for use in therapy, said population comprising myeloid cells or progenitor cells thereof that are designed to increase the cellular level of cellular FLICE inhibitor protein [Fas-associated death domain (FADD) similar to IL-1 p] converting enzyme (CFLAR), to pharmaceutical populations that comprise a population of cells comprising myeloid cells or progenitor cells thereof that are designed to increase the cellular level of cellular FLICE inhibitor protein [associated death domain a Fas (FADD)-like converting enzyme IL-1p] (CFLAR) and a nucleic acid encoding CFLAR, or a pharmaceutical composition comprising said nucleic acid, for use in therapy.

Background

The evolution of the immune system resulted in a highly effective network in vertebrates based on two types of defense: innate and adaptive immunity. Unlike the ancient evolutionary innate immune system that relies on invariant receptors that recognize common molecular patterns associated with pathogens, adoptive immunity relies on highly specific antigen receptors on B (B lymphocytes) and T (T lymphocytes) cells and on clonal selection. While B cells augment humoral immune responses by secreting antibodies, T cells mediate cellular immune responses leading to the destruction of recognized cells and play a major role in cell-mediated immunity in humans and animals.

Mature T cells recognize and respond to antigen through their antigen-specific receptors (TCR) that interact with immunogenic peptides (epitopes) bound to major histocompatibility complex (MHC) molecules and presented on the surface of target cells. For example, cytotoxic T cells respond to an antigen presented in association with MHC-I proteins. Helper T cells recognize antigen presented on MHC-II proteins. The most immediate consequence of TCR activation is the initiation of signaling pathways that result in clonal expansion of T cells, upregulation of activation markers on the cell surface, differentiation into effector cells, induction of cytotoxicity or secretion of cytokines and the induction of apoptosis. The TCR is part of a complex signaling machinery, which includes the heterodimeric complex of the TCR a and p chains, the CD4 or CD8 coreceptor, and the CD3 signal transduction module. While the CD3 chains transfer the activation signal into the cell, the a/p heterodimer of the TCR is solely responsible for antigen recognition.

In addition to the critical roles that T cells play in the immune response, dendritic cells (DCs) are equally important. DCs are professional antigen-presenting cells that play a key regulatory role in maintaining tolerance to self-antigens and activating innate and adaptive immunity against foreign antigens.

By producing soluble factors such as cytokines, interleukins and metabolites, tumors induce alternative hematopoiesis that modifies the normal differentiation of myeloid cells, driving the proliferation and expansion of cells with immunosuppressive functions called myeloid-derived suppressor cells (MDSC).Ugel S et al., 2015, J Clin Invest. 125:3365-76; Marvel D et al., 2015, J Clin Invest. 125:3356-64). Distinctive characteristics of MDSCs are myeloid origin, heterogeneous cellular composition, and the ability to negatively regulate adaptive and innate immune responses. The functional activity of MDSC is related to the upregulation of immunosuppressive enzymes, such as arginase 1 (ARG1), indoleamine 2,3-dioxygenase 1 (IDO1), NADPH oxidase (NOX), and inducible nitric oxide synthase (iNOS, also known as NOS2), as well as increased production of nitric oxide (NO) and reactive oxygen and nitrogen species (ROS and RNS), such as the free radical peroxynitrite (ONOO') (Bronte V et al., 2005, Nat Rev Immunol. 5:641-54) that inhibits the vitality and fitness of T cells. The composition of MDSCs is flexible and peculiar to each type of cancer and often changes, following the kinetics and development of the pathology: activated MDSCs They are mainly present in the primary tumor and metastatic sites, but can also be found in the circulation, suggesting that MDSC-specific activation markers could be used to differentiate them from other non-suppressive myeloid cells. (Gabrilovich DI et al., 2012, Nat Rev Immunol. 12:253-68; De Sanctis F et al, 2016, Biochim Biophys Acta. 1865:35-48). To date, human MDSCs have been classified into three main subsets: monocytic MDSCs (MO), polymorphonuclear/granulocytic MDSCs (PMN), and (i)-immature MDSCs. However, differences in sample preparation procedures and analysis of cytofluorimetric data, together with the lack of markers unique to MDSC, have led to many discrepancies between studies, slowing down the enumeration of MDSC in clinical routine. On the contrary, in the last twenty years, the identification of molecular and biological pathways related to MDSCs and the partial elucidation of cellular networks in the tumor microenvironment, through the use of innovative tumor mouse models, have generated greater interest in MDSCs and their role in cancer pathobiology. Recently, it has been reported that the heterogeneity of mouse MDSCs between the two major subsets is due to different activation of molecular pathways that regulate the death-inducing signaling complex (DISC). The generation of monocytic (MO)-MDSCs constitutively requires the presence of the antiapoptotic molecule cellular FLICE-inhibiting protein [Fas-associated death domain (FADD)-like IL-1 p-converting enzyme] (CFLAR, also known as c-FLIP )] that controls the extrinsic apoptotic pathway and the activation of caspase 8. On the other hand, the expansion of polymorphonuclear (PMN)-MDSCs depends on the MCL-1-mediated control of the intrinsic mitochondrial death pathway (Haverkamp JM et al. al., 2014, Immunity 41:947-59). CFLAR has 13 different splice variants, three of which are carried as proteins: the 26 kDa short form (c-FLIPs), the 24 kDa Raji form (c-FLIP<r>, specifically expressed only in some cell lines and human primary T cells) and the long 55 kDa form (c-FLIP<l>). All three CFLAR variants contain two death effector domains (DEDs) that interact with the apoptotic adapter protein FAAD. The gene that encodes CFLAR is evolutionarily conserved in vertebrates and the structure of c-FLIPS is similar to the viral FLICE inhibitory protein (v-FLIP) that is a component of viruses of the gammaherpesvirus class, such as human herpesvirus 8, a virus associated with Kaposi's sarcoma (Safa AR, 2012, Exp Oncol. 34:176-84; Safa AR et al., 2008, Curr Cancer Drug Targets 8:37-46). Wu et al., Cell death & differentiation 2013, 21: 451-461 describes THP-1 cells that overexpress<c>-FLIP<l>, Telieps et al., European Journal of Immunology 2013, 43: 1499-1510 describes mice transgenics that overexpress<c>-FLIP<r>in hematopoietic cells.

The immune system can also produce undesirable effects. For example, autoimmune disorders are characterized by loss of tolerance against self-antigens, activation of lymphocytes reactive against “self” antigens (autoantigens), and pathological damage to target organs. Additionally, the immune system can cause rejection of transplants of cells, tissues, and organs from unrelated donors. The immune system does not distinguish beneficial intruders, such as a transplanted tissue, from those that are harmful and therefore rejects the transplanted tissues or organs. Rejection of transplanted organs is generally mediated by alloreactive T cells present in the host that recognize donor alloantigens or xenoantigens. On the other hand, graft-versus-host disease can occur when immunologically active allogeneic cells are introduced, transplanted, or grafted into a host, resulting in a severe, even fatal, reaction due to immunological incompatibility between the host and the graft, where immunocompetent cells of the graft immunologically attack the host

Therefore, there is a need for agents that have tolerogenic or immunosuppressive actions. In particular, there is a need for agents that improve tolerogenicity in a host.

Description

Summary

The present teaching describes compositions, methods and uses for inducing immunosuppression and tolerance defined by the suppression of an immune response to an antigen.

The present invention is defined and limited by the attached claims.

The present invention is based on the concept that increasing the cellular level of cellular FLICE inhibitory protein [Fas-associated death domain (FADD) similar to IL-1 p-converting enzyme] (CFLAR) in cells, in particular myeloid cells or progenitor cells, increases the immunosuppressive activity of the cells. The cells in which the cellular level of CFLAR increases may be present, in vitro, that is, the cells may be isolated cells, or in vivo, that is, the cells may be present within a subject or individual. Therefore, the present teaching provides a method to enhance the tolerogenic or immunosuppressive potential of cells, in particular myeloid cells or their progenitor cells.

The tolerogenic or immunosuppressive cells described herein are useful for prolonging foreign graft survival in a mammalian host and for ameliorating inflammation-related diseases, such as autoimmune diseases. Tolerogenic or immunosuppressive cells are also useful for suppressing an immune response in the context of gene therapy of a desired gene or therapy based on exogenous proteins. Accordingly, the present invention encompasses compositions for reducing and/or eliminating an immune response to a transplant in a recipient by treating the recipient with an amount of tolerogenic or immunosuppressive cells described herein to reduce or inhibit rejection of the transplant by part of the guest. Transplantation refers to any material that is to be administered to a host. For example, a transplant includes, but is not limited to, a biocompatible network, a tissue, an organ, a cell, a nucleic acid, and a polypeptide. Also included are compositions for reducing and/or eliminating an immune response in a host by foreign versus host transplantation, i.e., graft versus host disease, by treating the transplant donor and/or the transplant recipient with tolerogenic cells. or immunosuppressives described herein in order to inhibit or reduce an adverse response of the transplanted donor against the recipient. Furthermore, the present invention encompasses compositions for reducing and/or eliminating an immune response to an exogenously administered protein in a recipient by treating the recipient with a number of tolerogenic or immunosuppressive cells described herein to reduce or inhibit rejection of the protein.

Various aspects of the present invention provide for the genetic modification of cells, either in vitro or in vivo, to express CFLAR or to express increased levels of CFLAR. Different vector formats can be used for genetic modification of cells, such as RNA, DNA or viral vectors that can be applied either in vitro or in vivo. If cells in which the cellular level of CFLAR is increased are present in vitro, strategies may depend on genetic modification of the cells in vitro and transferring the cells to a recipient after optional expansion of the cells in vitro. The cells used for treatment can be autologous, allogeneic or syngeneic of a recipient. If cells in which the cellular level of CFLAR is increased are present in vivo, genetic engineering can be performed, such as by transfection with nucleic acid of the cells in place in the patient, and strategies may depend on genetic modification of the cells. cells in vivo by administering an appropriate nucleic acid to a recipient, optionally in an appropriate vehicle.

Strategies for inducing immunosuppression and/or tolerance described herein can be used to suppress unwanted anticipated immune responses, for example, to prolong gene therapy or recurrent administration of therapeutic proteins, for example, expressed in a mammalian host by a vector or exogenously applied to a mammalian host, to prolong foreign graft survival in a host, to treat or prevent graft-versus-host disease, to treat or prevent autoimmune diseases, including, but not limited to, autoimmune arthritis, autoimmune diabetes, asthma, septic shock, pulmonary fibrosis, glomerulonephritis and atherosclerosis.

In one aspect, the invention relates to a population of cells for use in therapy, said population comprising myeloid cells or progenitor cells thereof that are designed to increase the cellular level of cellular FLICE [Fas-associated death domain] inhibitory protein. (FADD) similar to IL-1 converting enzyme p] (CFLAR), wherein said population comprises myeloid cells or progenitor cells thereof that comprise a recombinant nucleic acid encoding CFLAR, wherein the therapy comprises treating a patient requiring immunosuppression or in which the therapy comprises the treatment of a transplant recipient or graft versus host disease or an inflammatory disease or an immune disease. For example, the cellular level of CFLAR can be increased by increasing the expression of CFLAR in cells.

In one embodiment, increasing the cellular level of CFLAR increases the immunosuppressive activity of the cells.

In one embodiment, myeloid cells or their progenitor cells are engineered to increase cellular expression of CFLAR.

According to the invention, myeloid cells or their progenitor cells are transfected with nucleic acid encoding CFLAR. In one embodiment, CFLAR expression is increased in transfected cells compared to non-transfected cells. In one embodiment, the transfection is a stable or transient transfection. In one embodiment, myeloid cells or their progenitor cells are transfected using viral vectors as carriers, such as lentiviral vectors. In one embodiment, said nucleic acid is RNA.

In one embodiment, the CFLAR is CFLAR<l>and/or CFLARs.

In one embodiment, the myeloid cells or their progenitor cells are isolated and/or purified cells, in particular isolated and/or purified by magnetic cell sorting.

In one embodiment, the myeloid cells or their progenitor cells are monocytes. In one embodiment, the myeloid cells or progenitor cells thereof are CD14+ monocytes. In one embodiment, the myeloid cells or progenitor cells thereof are CD14+ monocytes isolated from buffy coats.

In one embodiment, the myeloid cells or their progenitor cells are stem cells. In one embodiment, the myeloid cells or their progenitor cells are bone marrow CD34+ cells. In one embodiment, the myeloid cells or progenitor cells thereof are bone marrow CD34+ cells isolated from bone marrow mononuclear cells (MNC).

In one embodiment, the cells have been treated to promote the acquisition of immunosuppressive activity. In one embodiment, treatment to promote the acquisition of immunosuppressive activity comprises in vitro differentiation in the presence of G-CSF and GM-CSF.

In one embodiment, the cell population of the invention also comprises myeloid cells or progenitor cells thereof that are not designed to increase the cellular level of CFLAR. In one embodiment, the cell population of the invention only comprises myeloid cells or progenitor cells thereof that are designed to increase the cellular level of CFLAR. In one embodiment, the cell population of the invention comprises cells other than myeloid cells or their progenitor cells. In one embodiment, the cell population of the invention only comprises myeloid cells or progenitor cells thereof.

In a further aspect, the invention relates to the population of cells described herein for use in therapy. In one embodiment, the therapy requires immunosuppression.

In a further embodiment, the invention relates to the population of cells described herein for treating a patient in need of immunosuppression. In one embodiment, the cell population is autologous to the patient. In a further aspect, the invention relates to a pharmaceutical composition for use in therapy, said pharmaceutical composition comprising the cell population described herein, wherein the therapy comprises treating a patient in need of immunosuppression or wherein the Therapy comprises the treatment of a transplant recipient or graft versus host disease or an inflammatory disease or an immune disease. A pharmaceutical composition of the invention may comprise a pharmaceutically acceptable carrier and may optionally comprise one or more adjuvants, stabilizers, etc. In a further aspect, the invention relates to a pharmaceutical composition described herein for use in therapy. In one embodiment, the therapy requires immunosuppression.

In a further embodiment, the invention relates to a pharmaceutical composition described herein for treating a patient in need of immunosuppression. In one embodiment, the cell population is autologous to the patient.

The present disclosure relates to a use of the cell population described herein for the production of a medicament for therapy, preferably the therapy requiring immunosuppression.

The present disclosure relates to a use of the cell population described herein for the production of a medicament to treat a patient in need of immunosuppression, preferably the cell population is autologous to the patient.

The present disclosure relates to a method of treating a patient in need of immunosuppression comprising administering the cell population described herein, preferably the cell population is autologous to the patient.

The present disclosure relates to a method for treating a patient in need of immunosuppression comprising increasing the cellular level of CFLAR in myeloid cells or progenitor cells thereof from the patient.

In one embodiment, increasing the cellular level of CFLAR increases the immunosuppressive activity of the cells.

In one embodiment, increasing the cellular level of CFLAR in the patient's myeloid cells or progenitor cells thereof comprises administering a nucleic acid encoding CFLAR. In one embodiment, the nucleic acid encoding CFLAR transfects myeloid cells or progenitor cells thereof. In one embodiment, said transfection is a stable or transient transfection. In one embodiment, myeloid cells or their progenitor cells are transfected using viral vectors as carriers, such as lentiviral vectors. In one embodiment, said nucleic acid is RNA.

In one embodiment, the CFLAR is CFLAR<l>and/or CFLARs.

In one embodiment, the myeloid cells or their progenitor cells are monocytes. In one embodiment, the myeloid cells or their progenitor cells are CD14+ monocytes.

In one embodiment, the myeloid cells or their progenitor cells are stem cells. In one embodiment, the myeloid cells or their progenitor cells are bone marrow CD34+ cells.

In a further aspect, the invention relates to a nucleic acid encoding CFLAR for use in therapy. In one embodiment, the therapy requires immunosuppression.

In a further embodiment, the invention relates to a nucleic acid encoding CFLAR for treating a patient in need of immunosuppression.

In a further aspect, the invention relates to a pharmaceutical composition comprising a nucleic acid encoding CFLAR for use in therapy.

In a further aspect, the invention relates to a pharmaceutical composition described herein for use in therapy. In one embodiment, the therapy requires immunosuppression.

In a further embodiment, the invention relates to a pharmaceutical composition described herein for treating a patient in need of immunosuppression.

The present disclosure relates to the use of a nucleic acid encoding CFLAR for the production of a medicament for therapy, preferably the therapy requiring immunosuppression.

The present disclosure relates to the use of a nucleic acid encoding CFLAR for the production of a medicament to treat a patient in need of immunosuppression.

In one embodiment of all aspects described herein, a therapy requiring immunosuppression is a transplant and a patient requiring immunosuppression is a transplant recipient. Thus, the invention also encompasses the population of cells, pharmaceutical composition or nucleic acid for use in the treatment of a transplant recipient to reduce in the recipient an immune response against the transplant and/or to treat or prevent rejection of the transplant by of the receiver. In different embodiments, the transplant is selected from the group consisting of a tissue, an organ, a cell, a nucleic acid, a protein, a biocompatible network and any combination thereof. In some aspects, the treatments described herein to induce immunosuppression and tolerance are administered to the recipient prior to transplantation. In other aspects, the treatments described herein to induce immunosuppression and tolerance are administered to the recipient simultaneously with the transplant. In still other aspects, the treatments described herein to induce immunosuppression and tolerance are administered as part of the transplant. In yet another aspect, the treatments described herein to induce immunosuppression and tolerance are administered to the recipient after transplantation.

In one embodiment of all aspects described herein, a therapy requiring immunosuppression is the treatment or prevention of graft-versus-host disease and a patient requiring immunosuppression is a patient who has graft-versus-host disease or who is in risk of developing graft-versus-host disease.

In one embodiment of all aspects described herein, a therapy requiring immunosuppression is the treatment or prevention of an immune disease and a patient requiring immunosuppression is a patient who has an immune disease or is at risk of developing an immune disease. .

In one aspect, the invention provides the agents and compositions such as cells and pharmaceutical compositions described herein for use in the treatment methods described herein.

In one aspect, the invention provides a viral vector comprising nucleic acid encoding CFLAR. In one embodiment, the viral vector is a lentiviral vector. The viral vector is preferably useful for engineering or transfecting myeloid cells or progenitor cells thereof in vivo or in vitro.

Other features and advantages of the present invention will become apparent from the following detailed description and claims.

Brief description of the drawings

Figure 1 shows the genetic sequence of long CFLAR (referred to as CFLAR<l>) with the plasmid used to generate the lentivirus encoding CFLAR<l>(A) and the sequence of the short CFLAR gene (referred to as CFLAR) with the plasmid used to generate the lentivirus that encodes CFLARs.

Figure 2 shows the protocol and results of immunosuppressive assays of infected human CD34+ cells expressing CFLAR and CFLARs to inhibit activated T cells compared to luciferase-expressing CD34+ cells used as a negative control (A), along with protocol and results of immunosuppressive assays of infected human CD14+ cells expressing CFLARL and CFLARs to inhibit activated T cells compared to CD14+ cells expressing luciferase used as negative control (B).

Figure 3 shows the heat map of up- and downregulated genes mediated by CFLAR-imposed expression in CD14+ cells compared to luciferase-infected CD14+ cells. CD14+ cells were obtained from four different buffy coats from healthy donors (HD).

Figure 4 shows the list of genes significantly upregulated by lentivirus-induced CFLAR expression in CD14+ cells compared to luciferase-infected CD14+ cells.

Figure 5 shows the validation of some gene targets modulated by forced CFLAR expression in CD14+ cells: upregulation of CFLARs, CFLAR<l>and IDOl genes by real-time PCR (A); IL-10 production by ELISA assay (B); and enhanced expression of surface markers CD273, CD274, CD80, CD163, CD44, CD38, and CD124 by flow cytometry.

Figure 6 shows the protocol (A) for treating mice subjected to GVHD grafted with human PBMC (the immunomodulatory approach was based on repeated administration of CFLAR-expressing monocytes) and the results of this treatment in prolonging the survival of the mice. treated compared to mice treated with non-transduced monocytes or transduced with a vector encoding luciferase (used as control); (B), pathological evaluation by histopathological scoring (C, upper panels); and enumeration of tissue-infiltrating human lymphocytes (defined as hCD3+ cells) by immunohistochemistry (C, lower panels).

Figure 7 shows the immune evaluation of mice subjected to GVHD treated with human CD14 + cells expressing CFLAR or luciferase as reported in protocol outline (A): mice treated with cells expressing CFLAR have a lower frequency of T cells hCD3+ circulating in the blood (B, panel i), spleen (B, panel ii) and bone marrow (B, iii); Furthermore, mice treated with CFLAR-expressing CD14+ cells have a lower frequency of hCD8+ T cells releasing IFN-<y>compared to mice receiving luciferase-expressing CD14+ cells after in vitro stimulation with PMA (50 ng /ml) plus ionomycin (1 mg/ml); Finally, mice treated with repeated injection of CFLAR-expressing CD14+ cells showed increased numbers of regulatory T cells (Treg) compared to mice treated with luciferase-expressing CD14+ cells.

Figure 8 shows the results of the immunosuppressive assay of CD11B+ Ly6G- Ly6Calto monocytes (panel A) and CD11B+ Ly6G+ Ly6Clow granulocytes (panel B) isolated from Rosa26.vFLIP;LysMcre (in black) derived from both bone marrow and spleen in comparison with CD11B+ Ly6G- Ly6Calto cells isolated from control, Rosa26.vFLIP (blank) mice.

Figure 9: c-FLAR induces an immunosuppressive program independently of its anti-apoptotic functions. a) Evaluation of the in vitro vitality of CD14+ cells infected with luciferase (as a control) or lentiviruses expressing c-FLAR (monocytes isolated from 4 different buffy coats). b) Only forced expression of c-FLAR induced immunosuppressive functions. In fact, monocytes infected with lentiviruses expressing BCL-2 or BCL did not show immunosuppressive capacity. All these data demonstrated that CFLAR directs immunosuppressive functions independently of protection against apoptosis.

Figure 10: Forced expression of c-FLAR in monocytes drives MDSC-associated molecular programs. a) Enrichment of GO BP terms for the 486 unique genes upregulated in c-FLAR overexpressed samples. Enrichment was determined using the GO over-representation test implemented in the Profiler group and GO Biological Process categories, p values were corrected using the BH procedure. b) Enrichment of signaling pathways for genes upregulated in samples overexpressed with c-FLAR (Figures 3, 4a and 4b). Differentially expressed genes were identified using SAM. Setting a q value <1% resulted in 486 unique genes upregulated in c-FLAR overexpressed samples (c-FLAR signature). Enrichment was determined using a Fisher test on all 267 GSEA signaling pathways. P values were corrected using the BH procedure. FDR q-val, false discovery rate q value.

Figure 11: c-FLAR activates the canonical NF-<k>B pathway in myeloid cells. a) THP1 cells transfected with c-FLAR RNA acquired immunosuppressive functions compared to the control (THP1 cells transfected with GFP-encoding RNA). Suppressive activity was measured by enumerating the absolute number of CD3+ T cells collected after co-culture with transfected THP1 cells. Data are presented as the mean ± SEM of three independent experiments. b) Immunofluorescence confocal microscopy of nuclear translocation of p65, p50 and p52 in transfected THP1 cells. Nuclei were counterstained with DAPI and slides were visualized using confocal microscopy. Original images at x800 for all panels. c) Ly6C+ cells expressing v-FLIP were purified and transfected with IKKa and IKKp siRNA and randomized for at least 18 hours. Myeloid cells were then incubated with Cell-Trace-labeled OT-I cells and co-cultured in the presence of SIINFEKL peptide for three days. Suppressive activity was measured by enumerating the absolute number of CD8+ T cells collected after co-culture. Data are presented as the mean ± SEM of four independent experiments.

Detailed description of the invention

Although the present invention is described in detail below, it should be understood that this invention is not limited to the particular methodologies, protocols and reagents described herein, as these may vary. It should also be understood that the terminology used herein is intended to describe particular embodiments only and is not intended to limit the scope of the present invention, which will be limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by one skilled in the art.

The elements of the present invention will be described below. These items are listed with specific embodiments; however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The various examples described and preferred embodiments should not be construed as limiting the present invention only to the explicitly described embodiments. This description should be understood to support and encompass embodiments that combine the explicitly described embodiments with any number of the disclosed and/or preferred elements. Furthermore, any permutation and combination of all elements described in this application should be deemed to be disclosed by the description of this application unless the context indicates otherwise.

Preferably, the terms used herein are defined as described in “A multilingual glossary of biotechnological terms: (IUPAC Recommendations)”, H.G.W. Leuenberger, B. Nagel and H. Kolbl, Eds., (1995) Helvetica Chimica Acta, CH-4010 Basel, Switzerland.

The practice of the present invention will employ, unless otherwise indicated, conventional methods of biochemistry, cell biology, immunology and recombinant DNA techniques that are explained in the literature in the field (see, for example, Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

Throughout this specification and the claims that follow, unless the context otherwise requires, the word “comprises” and variations such as “comprises” and “comprising” will be understood to imply the inclusion of a member, integer or indicated stage or group of members, integers or stages but without the exclusion of any other member, integer or stage or group of members, integers or stages although in some embodiments said other member, integer or stage or group of members, integers or stages They can be excluded, that is, the matter consists of the inclusion of a certain member, integer or stage or group of members, integers or steps. The terms “a” and “an” and “the” and similar references used in the context of the description of the invention (especially in the context of the claims) should be construed to cover both the singular and the plural, unless otherwise specified. indicates otherwise herein or clearly contradicted by the context.

The enumeration of value ranges in this document is simply intended to serve as a shorthand method for referring individually to each separate value that falls within the range. Unless otherwise noted herein, each individual value is incorporated into the specification as if it were individually listed herein.

All methods described herein may be performed in any suitable order unless otherwise indicated herein or clearly contradicted by the context. The use of any and all examples, or exemplary language (e.g., “as”), provided herein is intended simply to better illustrate the invention and does not represent a limitation on the scope of the claimed invention. No language in the specification should be construed as indicating any unclaimed element essential to the practice of the invention.

The term "recombinant" in the context of the present invention means "produced by genetic engineering." Preferably, a "recombinant object", such as a recombinant cell or nucleic acid in the context of the present invention, does not occur naturally.

The term “naturally occurring,” as used herein, refers to the fact that an object can be found in nature. For example, a peptide or nucleic acid that is present in an organism (including viruses) and that can be isolated from a source in nature and that has not been intentionally modified by man in the laboratory is of natural origin.

As used herein, an “isolated” cell is a cell that is essentially free of other cell types. An isolated cell also refers to a cell that has been separated from other cell types with which it is normally associated in its natural state. In some cases, a population of isolated cells refers to a homogeneous population of cells. In other cases, this term simply refers to cells that have been separated from the cells with which they are naturally associated in their natural state. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro. In one embodiment, a cell population described herein contains 50% or more of the desired cells in the cell population, preferably 75% or more of the cell population, more preferably 85% or 90% or more of the cell population, and most preferably 95% or more of the cell population.

As used herein, a “purified” cell is a cell that is essentially free of unwanted components such as cellular material and/or proteins contaminating the tissue source from which the cell is derived. A purified cell also refers to a cell that has been separated from unwanted components.

In one embodiment of the invention, a cell population comprising myeloid cells or progenitor cells thereof refers to a cell population that is substantially free of cells other than myeloid cells or progenitor cells thereof and/or non-myeloid components. desired.

As the term is used herein, “separated from” or “separates” refers to the characteristic that a population of first substances is removed from the proximity of a population of second substances, in which the population of first substances substances is not necessarily devoid of the second substance, and the population of second substances is not necessarily devoid of the first substance. However, a population of first substances that is “separated from” a population of second substances has a measurably lower content of second substances compared to the unseparated mixture of first and second substances.

Hematopoietic blood cells are derived from pluripotent bone marrow stem cells through proliferation and differentiation. Stem cells, which are characterized by their repertoire of cell surface receptors (e.g., CD34+), are undifferentiated. Through rounds of self-renewal and expansion by proliferation, followed by differentiation in response to cytokines and growth factors, these cells give rise to all mature functional blood cells (monocytes/macrophages, eosinophils, neutrophils, T lymphocytes, and B lymphocytes).

In blood cell differentiation, two lineage pathways are clearly delineated; myeloid (including monocytes/macrophages, eosinophils and neutrophils) and lymphoid (including T lymphocytes and B lymphocytes). Broadly speaking, myeloid cells perform functional roles associated with the rapid destruction and elimination of pathogens (such as bacteria and parasites), while lymphoid cells are associated with the memory of pathogens and the generation of an antibody-mediated immune response. Myeloid cells perform a more transient function and are therefore produced in large numbers, survive days/weeks rather than months or years, and are fully specialized and terminally differentiated functional cells that do not divide but remain in the phase. GO/GI of the cell cycle. Once they have performed their function, they die by apoptosis and are eliminated by phagocytosis. In contrast, and due to their specialized memory function, T and B lymphocytes can live a long time and are capable of extensive proliferation and cell cycle.

Therefore, the term “myeloid cells” designates cells of myeloid origin, encompasses terminally differentiated, nondividing (i.e., nonproliferative) cells derived from the myeloid cell lineage, and includes neutrophils or polymorphonuclear neutrophils (PMNs), eosinophils, and phagocytes. mononuclear. These latter cells are known as monocytes when they are in the blood and macrophages when they have migrated to the tissues. Terminal differentiation is the normal endpoint of cellular differentiation and is normally not reversible.

In a particularly preferred embodiment, the term "myeloid cells" refers to monocytes. The term “monocyte,” as used herein, refers to white blood cells that circulate in the bloodstream. Monocytes are found in circulating peripheral blood, where they constitute 10 to 20% of the total mononuclear cell population. Monocytes are characterized by their phenotypic expression of CD14 and are therefore commonly called CD14+ monocytes. They play an important role in host defense as circulating monocytes and can differentiate into tissue macrophages and dendritic cells with potent antigen-presenting capacity. Monocytes differentiate into macrophages upon migrating into tissues. Therefore, as used herein, the term “monocyte” also refers to macrophages.

The term “buffy coat” refers to the fraction of an anticoagulated blood sample that contains the majority of white blood cells and platelets after density gradient centrifugation of the blood.

Myeloid cell progenitor cells include hematopoietic cells such as hematopoietic stem cells and myeloid progenitor cells. In particular, myeloid cell progenitor cells include cells that express CD34+ (CD34+ cells). CD34-expressing cells are normally found in the umbilical cord and bone marrow as hematopoietic cells. Bone marrow CD34+ cells contain hematopoietic stem and progenitor cells and are known to differentiate into all different types of blood cells. CD34+ cells can be isolated from blood samples using immunomagnetic or immunofluorescent methods.

Terms such as “reduce”, “inhibit” or “decrease” relate to the ability to cause an overall decrease, preferably of 5% or more, 10% or more, 20% or more, more preferably 50% or more, and most preferably 75% or more, in level. These terms include complete or essentially complete inhibition, that is, a reduction to zero or essentially zero.

Terms such as “increase”, “improve” or “promote” relate to the ability to cause an overall increase, preferably 5% or more, 10% or more, 20% or more, 50% or more, 75% or more, 100% or more, 200% or more, or 500% or more at the level. These terms may relate to an increase, improvement or promotion from zero or a non-measurable or non-detectable level to a level greater than zero or a level that is measurable or detectable. Alternatively, these terms can also mean that there was a certain level before a raise, upgrade or promotion and after the raise, upgrade or promotion the level is higher.

According to the present invention, the term "increasing the cellular level" with respect to a particular substance such as CFLAR refers to measures that result in a greater degree or amount of the substance compared to the normal situation, in particular the normal situation in a cell, in which the cellular level has not increased/has not been increased by man.

The term "immune response" refers to an integrated bodily response to an antigen and preferably refers to a cellular immune response, a humoral immune response, or both. According to the invention, the term "immune response to" or "immune response against" with respect to an agent such as an antigen, cell or tissue, refers to an immune response such as a cellular response directed against the agent.

The terms “cellular immune response”, “cellular response”, “cell-mediated immunity” or similar terms are intended to include a cellular response directed at cells characterized by the expression of an antigen and/or the presentation of an antigen with class I or class II MHC. The cellular response is related to cells called T cells or T lymphocytes that act as “helpers” or “killers.” Helper T cells (also called CD4+ T cells) play a major role in regulating the immune response and killer cells (also called cytotoxic T cells, cytolytic T cells, CD8+ T cells or CTL) destroy cells such as diseased cells.

The term “humoral immune response” refers to a process in living organisms in which antibodies are produced in response to agents and organisms, which they ultimately neutralize and/or eliminate. The specificity of the antibody response is mediated by T and/or B cells through membrane-associated receptors that bind antigens of a single specificity. Following binding of an appropriate antigen and receipt of several other activating signals, B lymphocytes divide, producing memory B cells as well as clones of antibody-secreting plasma cells, each of which produces antibodies that They recognize the antigenic epitope identical to that recognized by their antigen receptor. Memory B lymphocytes remain dormant until they are later activated by their specific antigen. These lymphocytes provide the cellular basis for memory and the subsequent intensification of the antibody response when re-exposed to a specific antigen.

The term “antibody,” as used herein, refers to an immunoglobulin molecule that is capable of specifically binding to an epitope of an antigen. In particular, the term "antibody" refers to a glycoprotein comprising at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. The term "antibody" includes monoclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, chimeric antibodies and combinations of any of the above. Each heavy chain is composed of a heavy chain variable (VH) region and a heavy chain constant (CH) region. Each light chain is composed of a variable light chain (VL) region and a constant light chain (CL) region. Variable regions and constant regions are also referred to here as variable domains and constant domains, respectively. The VH and VL regions can be subdivided into regions of hypervariability, called complementarity determining regions (CDR), interspersed with regions that are more conserved, called framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs of a VH are called HCDR1, HCDR2 and HCDR3, the CDRs of a VL are called LCDR1, LCDR2 and LCDR3. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of an antibody comprise the heavy chain constant region (CH) and the light chain constant region (CL), in which CH can be further subdivided into constant domain CH1, a hinge region, and constant domains CH2 and cH3 (arranged from amino-terminal to carboxy-terminal in the following order: CH1, CH2, CH3). Constant regions of antibodies may mediate immunoglobulin binding to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The antibodies may be intact immunoglobulins derived from natural sources or from recombinant sources and may be immunoactive portions of intact immunoglobulins. Antibodies are usually tetramers of immunoglobulin molecules. Antibodies can exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)2, as well as single chain antibodies and humanized antibodies.

The term "immunoglobulin" refers to proteins of the immunoglobulin superfamily, preferably antigen receptors such as antibodies or the B cell receptor (BCR). Immunoglobulins are characterized by a structural domain, i.e. the immunoglobulin domain, which has a characteristic immunoglobulin (Ig) fold. The term encompasses membrane-bound immunoglobulins as well as soluble immunoglobulins. Membrane-bound immunoglobulins are also called surface immunoglobulins or membrane immunoglobulins, which are usually part of the BCR. Soluble immunoglobulins are usually called antibodies. Immunoglobulins generally comprise several chains, typically two identical heavy chains and two identical light chains that are linked by disulfide bonds. These chains are mainly composed of immunoglobulin domains, such as the V<l>domain (variable light chain), C<l>domain (constant light chain), V<l>domain (variable heavy chain), and the C<l>h >(constant heavy chain) domains C<h>1, C<h>2, C<h>3 and C<h>4. There are five types of mammalian immunoglobulin heavy chains, i.e., a, 5, £, y and M, which represent the different classes of antibodies, i.e., IgA, IgD, IgE, IgG and IgM. Unlike the heavy chains of soluble immunoglobulins, the heavy chains of membrane or surface immunoglobulins comprise a transmembrane domain and a short cytoplasmic domain at their carboxyl terminus. In mammals there are two types of light chains, that is, lambda and kappa. Immunoglobulin chains comprise a variable region and a constant region. The constant region is essentially conserved within the different immunoglobulin isotypes, while the variable part is very diverse and represents antigen recognition.

An "antigen" according to the invention covers any substance that provokes an immune response and/or any substance against which an immune response or an immune mechanism such as a cellular response is directed. This also includes situations where antigen is processed into antigenic peptides and an immune response or immune mechanism is directed against one or more antigenic peptides, particularly if presented in the context of MHC molecules. In particular, an "antigen" refers to any substance, preferably a peptide or protein, that specifically reacts with T lymphocytes (T cells). According to the present invention, the term "antigen" comprises any molecule that comprises at least one epitope such as a T cell epitope. Preferably, an antigen in the context of the present invention is a molecule that, optionally after processing, induces an immune reaction, which is preferably specific for the antigen (including cells that express the antigen). In the context of embodiments of the present invention, the antigen is preferably presented by a cell in the context of MHC molecules, resulting in an immune response against the antigen.

As used herein, the term “autoantigen” refers to an antigen that is native to a mammal and that may be involved in the pathogenesis of an immune disease.

The term “immunogenicity” refers to the relative effectiveness of an antigen in inducing an immune reaction.

The term “immunostimulator” is used herein to refer to the increase in the general immune response.

The term “immunosuppressant” is used herein to refer to the reduction of the overall immune response.

In some cases, it is desirable to induce an antigen-specific immunosuppressive effect.

“Immune tolerance”, “immunological tolerance” or simply “tolerance” describes a state of lack of response of the immune system to substances or tissues that have the capacity to provoke an immune response. It contrasts with conventional immune-mediated elimination of foreign antigens. The mechanisms by which tolerance is established are different, but the resulting effect is similar.

An "antigenic peptide" according to the invention preferably refers to a portion or fragment of an antigen that is capable of stimulating an immune response, preferably a cellular response against the antigen or cells characterized by the expression of the antigen and preferably by the presentation of the antigen. Preferably, an antigenic peptide is capable of stimulating a cellular response against a cell characterized by the presentation of an MHC class I antigen and preferably is capable of stimulating a cytotoxic T lymphocyte (CTL) sensitive to the antigen. Preferably, the antigenic peptides are MHC class I and/or class II presented peptides. Preferably, the antigenic peptides comprise an amino acid sequence that substantially corresponds to the amino acid sequence of a fragment of an antigen. Preferably, said fragment of an antigen is a peptide presented by MHC class I and/or class II.

A peptide that is suitable for binding to an MHC molecule, in particular an MHC class I molecule, preferably has a length of 7-20 amino acids, more preferably a length of 7-12 amino acids, more preferably a length of 8-12 amino acids. 11 amino acids in particular 9 or 10 amino acids in length.

In one embodiment, an antigenic peptide when presented in the context of MHC such as MHC of antigen-presenting cells is recognized by a T cell receptor. The antigenic peptide, if recognized by a T cell receptor, may be capable of induce, in the presence of appropriate costimulatory signals, the clonal expansion of the T cell carrying the T cell receptor that specifically recognizes the antigenic peptide. Preferably, the antigenic peptides, particularly if presented in the context of MHC molecules, are capable of stimulating an immune response, preferably a cellular response against the antigen from which they are derived or cells characterized by the expression of the antigen and preferably characterized by the presentation of the antigen.

The term "epitope" refers to an antigenic determinant on a molecule such as an antigen, that is, to a part or fragment of the molecule that is recognized by the immune system, for example, that is recognized by a T cell, in particularly when presented in the context of MHC molecules. An epitope of a protein preferably comprises a continuous or discontinuous portion of said protein and is preferably between 5 and 100, preferably between 5 and 50, more preferably between 8 and 30, most preferably between 10 and 25 amino acids in length, e.g. the epitope may preferably be 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids in length. It is particularly preferred that the epitope in the context of the present invention is a T cell epitope.

Terms such as "epitope", "T cell epitope", "fragment of an antigen", "immunogenic peptide" and "antigenic peptide" are used interchangeably herein and preferably relate to an incomplete representation of an antigen that is preferably capable of eliciting an immune response against the antigen or a cell that expresses or comprises and preferably presents the antigen. Preferably, the terms refer to an immunogenic portion of an antigen. Preferably, it is a portion of an antigen that is recognized (i.e., specifically bound) by a T cell receptor, particularly if presented in the context of MHC molecules. Certain preferred immunogenic portions bind to an MHC class I or class II molecule.

The term “target” will mean an agent such as a cell or tissue that is a target for an immune response such as a cellular immune response. Targets include cells that present an antigen or an antigenic epitope, that is, a peptide fragment derived from an antigen. In one embodiment, the target cell is a cell that expresses an antigen and preferably presents said antigen with MHC class I.

The term “portion” refers to a fraction. With respect to a particular structure such as an amino acid sequence or a protein, the term “portion” thereof may designate a continuous or discontinuous fraction of said structure.

The terms “part” and “fragment” are used interchangeably herein and refer to a continuous element. For example, a part of a structure such as an amino acid sequence or a protein refers to a continuous element of said structure.

“Antigen processing” refers to the degradation of an antigen into process products, which are fragments of said antigen (e.g., the degradation of a protein into peptides) and the association of one or more of these fragments (e.g., by binding) with MHC molecules for presentation by cells, preferably antigen-presenting cells to specific T cells.

An antigen-presenting cell (APC) is a cell that displays an antigen in the context of the major histocompatibility complex (MHC) on its surface. T cells can recognize this complex using their T cell receptor (TCR). Antigen-presenting cells process antigens and present them to T cells. An antigen-presenting cell includes, but is not limited to, monocytes/macrophages, B cells, and dendritic cells (DCs).

Nonprofessional antigen-presenting cells do not constitutively express MHC class II proteins necessary for interaction with naïve T cells; These are expressed only after the stimulation of non-professional antigen-presenting cells by certain cytokines such as IFNy.

Professional antigen-presenting cells are very efficient at internalizing the antigen, either by phagocytosis or receptor-mediated endocytosis, and then display a fragment of the antigen, bound to an MHC class II molecule, on their membrane. The T cell recognizes and interacts with the MHC class II antigen molecular complex on the membrane of the antigen-presenting cell. The antigen-presenting cell then produces an additional costimulatory signal, leading to T cell activation. Expression of costimulatory molecules is a defining characteristic of professional antigen-presenting cells.

The main types of professional antigen-presenting cells are dendritic cells, which have the widest range of antigen presentation and are probably the most important antigen-presenting cells, macrophages, B cells, and certain activated epithelial cells.

Dendritic cells (DCs) are populations of leukocytes that present antigens captured in peripheral tissues to T cells through MHC class II and I antigen presentation pathways. It is well known that dendritic cells are potent inducers of immune responses. and the activation of these cells is a critical step for the induction of immunity.

Dendritic cells are conveniently classified into “immature” and “mature” cells, which can be used as a simple way to discriminate between two well-characterized phenotypes. However, this nomenclature should not be interpreted as excluding all possible intermediate stages of differentiation.

Immature dendritic cells are characterized as antigen-presenting cells with a high capacity for antigen uptake and processing, which correlates with the high expression of the Fcy receptor and the mannose receptor. The mature phenotype is typically characterized by lower expression of these markers, but high expression of cell surface molecules responsible for T cell activation, such as MHC class I and class II adhesion molecules (e.g. , CD54 and CD11) and costimulatory molecules (for example, CD40, CD80, CD86 and 4-1 BB).

Dendritic cell maturation is known as the state of dendritic cell activation in which antigen-presenting dendritic cells lead to T cell priming, while presentation by immature dendritic cells results in tolerance. The maturation of dendritic cells is mainly caused by biomolecules with microbial characteristics detected by innate receptors (bacterial DNA, viral RNA, endotoxins, etc.), proinflammatory cytokines (TNF, IL-1, IFN), ligation of CD40 on the surface of dendritic cells by CD40L and substances released by cells undergoing stressful cell death. Dendritic cells can be derived by culturing bone marrow cells in vitro with cytokines, such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor-alpha.

The term "immunoreactive cell" or "effector cell" in the context of the present invention refers to a cell that exerts effector functions during an immune reaction. An "immunoreactive cell" is preferably capable of binding to an antigen or a cell characterized by the expression and/or presentation of an antigen or an antigenic peptide derived from an antigen and mediate an immune response. For example, said cells secrete cytokines and/or chemokines, destroy microbes, secrete antibodies, recognize infected or cancerous cells and, optionally, eliminate said cells. For example, immunoreactive cells comprise T cells (cytotoxic T cells, helper T cells, tumor-infiltrating T cells), B cells, natural killer cells, neutrophils, macrophages, and dendritic cells. Preferably, in the context of the present invention, “immunoreactive cells” are T cells, preferably CD4+ and/or CD8+ T cells.

Preferably, an "immunoreactive cell" recognizes an antigen or an antigenic peptide derived from an antigen with a certain degree of specificity, particularly if presented in the context of MHC molecules such as on the surface of antigen-presenting cells or diseased cells such as tumor cells. Preferably, said recognition allows the cell that recognizes an antigen or an antigenic peptide derived from said antigen to respond or be reactive. If the cell is a helper T cell (CD4+ T cells) containing receptors that recognize an antigen or an antigenic peptide derived from an antigen in the context of MHC class II molecules, such responsiveness or reactivity may involve the release of cytokines and/or the activation of CD8+ lymphocytes (CTL) and/or B cells. If the cell is a CTL, said responsiveness or reactivity may involve the elimination of cells presented in the context of MHC class I molecules, that is That is, cells characterized by the presentation of an MHC class I antigen, for example, through perforin-mediated cell lysis or apoptosis. According to the invention, the responsiveness of CTL may include sustained calcium flux, cell division, production of cytokines such as IFN-y and TNF-a, upregulation of activation markers such as CD44 and CD69, and death. specific cytolysis of target cells that express antigens. CTL responsiveness can also be determined using an artificial indicator that accurately indicates CTL responsiveness. Such CTLs that recognize an antigen or an antigenic peptide derived from an antigen and are responsive or reactive are also referred to herein as “antigen-responsive CTLs.” If the cell is a B cell, such responsiveness may involve the release of immunoglobulins.

The term “T cell” or “T lymphocyte” relates to cells derived from the thymus that participate in a variety of cell-mediated immune reactions and includes helper T cells (CD4+ T cells) and cytotoxic T cells (CTL, CD8+ T cells). comprising cytolytic T cells.

T cells belong to a group of white blood cells known as lymphocytes and play a major role in cell-mediated immunity. They can be distinguished from other types of lymphocytes, such as B cells and natural killer cells, by the presence of a special receptor on their cell surface called the T cell receptor (TCR). The thymus is the main organ responsible for T cell maturation. Several different T cell subsets have been discovered, each with a distinct function.

Helper T cells assist other white blood cells in immunological processes, including the maturation of B cells into plasma cells and the activation of cytotoxic T cells and macrophages, among other functions. These cells are also known as CD4+ T cells because they express the CD4 protein on their surface. Helper T cells are activated when presented with peptide antigens by MHC class II molecules that are expressed on the surface of antigen-presenting cells (APCs). Once activated, they divide rapidly and secrete small proteins called cytokines that regulate or assist in the active immune response.

Cytotoxic T cells destroy virus-infected cells and tumor cells, and are also involved in transplant rejection. These cells are also known as CD8+ T cells because they express the CD8 glycoprotein on their surface. These cells recognize their targets by binding to the MHC class I-associated antigen, which is present on the surface of almost all cells in the body.

Most T cells have a T cell receptor (TCR) that exists as a complex of several proteins. The actual T cell receptor is composed of two separate peptide chains, which are produced from the independent T cell receptor alpha and beta genes (TCRa and TCRp) and are called the a- and p-TCR chains. Y¿ T cells (gamma delta T cells) represent a small subset of T cells that possess a distinct T cell receptor (TCR) on their surface. However, in y§ T cells, the TCR is made up of a y chain and an 8 chain. This group of T cells is much less common (2% of total T cells) than ap T cells.

The structure of the T cell receptor is very similar to immunoglobulin Fab fragments, which are regions defined as the combined light and heavy chain of an antibody arm. Each TCR chain is a member of the immunoglobulin superfamily and possesses an N-terminal immunoglobulin (Ig) variable (V) domain, a constant Ig (C) domain, a transmembrane/cell membrane region, and a short cytoplasmic tail at the end of the chain. C terminal. The variable domain of both the a chain and the p chain of the TCR have three hypervariable complementarity determining regions (CDRs), while the variable region of the p chain has an additional area of hypervariability (HV4) that normally does not contacts the antigen and is therefore not considered a CDR. CDR3 is the main CDR responsible for the recognition of the processed antigen, although it has also been shown that CDR1 of the a chain interacts with the N-terminal part of the antigenic peptide, while CDR1 of the p chain interacts with the C-terminal part of the antigenic peptide. CDR2 is considered to recognize the MHC. CDR4 of the p chain is not considered to be involved in antigen recognition, but has been shown to interact with superantigens. The constant domain of the TCR domain consists of short connecting sequences in which a cysteine residue forms disulfide bonds, which form a bond between the two chains.

The term “peripheral blood mononuclear cell” or “PBMC” refers to a peripheral blood cell that has a round nucleus. These cells are made up of lymphocytes (T cells, B cells, NK cells) and monocytes, while erythrocytes and platelets do not have a nucleus and granulocytes (neutrophils, basophils and eosinophils) have multilobed nuclei. These cells can be extracted from whole blood using ficoll and gradient centrifugation, which will separate the blood into an upper layer of plasma, followed by a layer of PBMCs and a lower fraction of polymorphonuclear cells (such as neutrophils and eosinophils) and erythrocytes.

The term “major histocompatibility complex” and the abbreviation “MHC” include MHC class I and MHC class II molecules and refer to a gene complex found in all vertebrates. MHC proteins or molecules are important for signaling between lymphocytes and antigen-presenting cells or diseased cells in immune reactions, in which MHC proteins or molecules bind to peptides and present them for recognition by T cell receptors. MHC-encoded proteins are expressed on the surface of cells and display both self-antigens (fragments of the cell's own peptides) and foreign antigens (e.g., fragments of invading microorganisms) to a T cell.

The MHC region is divided into three subgroups, class I, class II and class III. MHC class I proteins contain an a chain and a p2 microglobulin (which is not part of the MHC encoded by chromosome 15). They present antigen fragments to cytotoxic T cells. In most cells of the immune system, specifically antigen-presenting cells, MHC class II proteins contain a and p chains and present antigen fragments to helper T cells. The MHC class III region encodes other immune components, such as complement components and some that encode cytokines.

In humans, genes in the MHC region that encode antigen-presenting proteins on the cell surface are called human leukocyte antigen (HLA) genes. However, the abbreviation MHC is often used to refer to HLA gene products. HLA genes include the nine so-called classical MHC genes: HLA-A, HLA-B, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA and HLA-DRB1.

In a preferred embodiment of all aspects of the invention, an MHC molecule is an HLA molecule.

The term “immune effector functions” or “effector functions” in the context of the present invention includes any function mediated by components of the immune system that results, for example, in the destruction of cells. Preferably, immune effector functions in the context of the present invention are effector functions mediated by T cells. Such functions comprise in the case of a helper T cell (CD4+ T cell) the recognition of an antigen or an antigenic peptide derived from an antigen. in the context of MHC class II molecules by T cell receptors, the release of cytokines and/or the activation of CD8+ lymphocytes (CTL) and/or B cells, and in the case of CTL, the recognition of an antigen or an antigenic peptide derived from an antigen in the context of MHC class I molecules by T cell receptors, the elimination of cells presented in the context of MHC class I molecules, that is, cells characterized by the presentation of an antigen with MHC class I, for example, through perforin-mediated cell lysis or apoptosis, production of cytokines such as IFN-γ and TNF-α, and specific cytolytic destruction of antigen-expressing target cells.

A "nucleic acid" according to the invention is preferably deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), more preferably RNA, most preferably in vitro transcribed RNA (IVT RNA) or synthetic RNA. Nucleic acids according to the invention include genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules. According to the invention, a nucleic acid can be present as a linear or circularly covalently closed single- or double-stranded molecule. According to the invention a nucleic acid can be isolated. The term "isolated nucleic acid" means, according to the invention, that the nucleic acid (i) was amplified in vitro, for example by polymerase chain reaction (PCR), (ii) was produced recombinantly by cloning , (iii) was purified, for example, by cleavage and separation by gel electrophoresis, or (iv) was synthesized, for example, by chemical synthesis. A nucleic acid can be used for introduction into cells, that is, transfection of them, for example, in the form of RNA that can be prepared by in vitro transcription from a DNA template. Furthermore, RNA can be modified before application by stabilizing, capping, and polyadenylation sequences.

The nucleic acids described herein may be comprised in a vector that can be used to deliver a nucleic acid into a cell. The term "vector", as used herein, includes any vector known to the person skilled in the art, including plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as adenoviral or baculoviral vectors, retro or lentiviral vectors, transposons or artificial chromosome vectors. such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC) or P1 artificial chromosomes (PAC). Such vectors include expression vectors as well as cloning vectors. Expression vectors comprise plasmids as well as viral vectors and generally contain a desired coding sequence and appropriate DNA sequences necessary for expression of the operably linked coding sequence in a particular host organism (e.g. bacteria, yeast, plants, insects or mammals). ) or in in vitro expression systems. Cloning vectors are generally used to engineer and amplify a certain desired DNA fragment and may lack functional sequences necessary for the expression of the desired DNA fragments.

The tolerogenic or immunosuppressive cells described herein can be generated either in vitro or in vivo by transfecting or transducing the cells with a vector that results in increased expression of CFLAR. Any of a variety of methods well known to one skilled in the art can be used to transfect or transduce the cells.

The nucleic acid encoding CFLAR can be cloned into several types of vectors. However, the present invention should not be considered to be limited to any particular vector. Instead, the present invention should be interpreted as encompassing a wide variety of vectors that are readily available and well known in the art. In specific embodiments, the vector is selected from the group consisting of a viral vector, a bacterial vector, and a mammalian cell vector. Many of these systems are commercially and widely available.

The vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art. Viruses that are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses and lentiviruses. Preferably, the virus is a helper-dependent adenovirus (HD-Ad). In general, a suitable vector contains a functional origin of replication in at least one organism, a promoter sequence, convenient restriction endonuclease sites and one or more selectable markers.

Those skilled in the art of molecular biology generally know how to use promoters, enhancers, and combinations of cell types for protein expression. The promoters employed may be constitutive, tissue-specific, inducible and/or useful under appropriate conditions to drive high-level expression of the introduced nucleic acid segment encoding CFLAR. The promoter can be heterologous or endogenous. Constitutive promoter sequences that can be used in accordance with the invention include, among others, the cytomegalovirus (CMV) immediate early promoter sequence, the simian virus 40 (SV40) early promoter, the mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR), Moloney virus promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, as well as promoters of human genes such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter and the muscle creatine promoter. Furthermore, the invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter in the invention provides a molecular switch capable of turning on expression of the polynucleotide sequence to which it is operably linked when such expression is desired, or turning off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter. Furthermore, the invention includes the use of a tissue-specific promoter, which promoter is active only in a desired tissue. Tissue-specific promoters are well known in the art and include, but are not limited to, the HER-2 promoter and PSA-associated promoter sequences.

To evaluate the expression of CFLAR, the expression vector to be introduced into a cell may also contain a selectable marker gene or a reporter gene or both to facilitate the identification and selection of expressing cells from the target cell population. transfect or infected through viral vectors. In other embodiments, the selectable marker can be carried on a separate piece of DNA and used in a cotransfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to allow expression in host cells. Selectable markers useful in the art are known and include, for example, antibiotic resistance genes, such as neo and the like. Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. Reporter genes that encode easily analyzed proteins are well known in the art. In general, a reporter gene is a gene that is not present or expressed in the recipient organism or tissue and that encodes a protein whose expression is manifested by some easily detectable property, for example, enzymatic activity. The expression of the reporter gene is analyzed at a suitable time after the nucleic acid has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase or the green fluorescent protein gene.

The vector can be easily introduced into a host cell by any method in the art. For example, the expression vector can be transferred to a host cell by physical, chemical or biological means. Physical methods for introducing a nucleic acid into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York).

Biological methods for introducing a nucleic acid of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, for example, human cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like.

Chemical means for introducing a nucleic acid into a host cell include colloidal dispersion systems, such as complexes of macromolecules, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. A preferred colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (i.e., an artificial membrane vesicle). The preparation and use of such systems is well known in the art.

Regardless of the method used to introduce exogenous nucleic acids into a host cell or otherwise increase the cellular level of CFLAR in a cell, in order to confirm the presence and/or amount of CFLAR or its encoding nucleic acid in the host cell, A variety of tests can be performed. Such assays include, for example, Southern and Northern blots, RT-PCR and PCR, and assays to detect the presence or absence of a particular peptide, for example, by immunological means (ELISA and Western blots).

In the context of the present invention, the term "DNA" refers to a molecule that comprises deoxyribonucleotide residues and preferably is composed wholly or substantially of deoxyribonucleotide residues. "Deoxyribonucleotide" refers to a nucleotide lacking a hydroxyl group at the 2' position of a p-D-ribofuranosyl group. The term "DNA" comprises isolated DNA such as partially or completely purified DNA, essentially pure DNA, synthetic DNA and recombinantly generated DNA and includes modified DNA that differs from natural DNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations may include the addition of non-nucleotide material, such as at the end(s) of a DNA or internally, for example at one or more nucleotides of the DNA. The nucleotides in DNA molecules may also comprise non-standard nucleotides, such as nucleotides of non-natural origin or chemically synthesized nucleotides. These altered DNAs can be called natural DNA analogs or analogues.

In the context of the present invention, the term "RNA" refers to a molecule that comprises ribonucleotide residues and is preferably composed entirely or substantially of ribonucleotide residues. "Ribonucleotide" refers to a nucleotide with a hydroxyl group at the 2' position of a p-D-ribofuranosyl group. The term includes double-stranded RNA, single-stranded RNA, isolated RNA such as partially or completely purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as modified RNA that differs from natural RNA by addition, deletion, substitution and/or or alteration of one or more nucleotides. Such alterations may include the addition of non-nucleotide material, such as at the end(s) of an RNA or internally, for example at one or more nucleotides of the RNA. Nucleotides in RNA molecules may also comprise non-standard nucleotides, such as nucleotides of non-natural origin or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be called natural RNA analogs or analogs. According to the present invention, the term "RNA" includes and preferably refers to "mRNA", which means "messenger RNA" and refers to a transcript that can be produced using DNA as a template and encodes a peptide or protein. The mRNA typically comprises a 5' untranslated region (5'-UTR), a protein or peptide coding region, and a 3' untranslated region (3'-UTR). mRNA has a limited half-time in cells and in vitro. Preferably, the mRNA is produced by in vitro transcription using a DNA template. In one embodiment of the invention, the RNA is obtained by in vitro transcription or chemical synthesis. The in vitro transcription methodology is known to the person skilled in the art. For example, there are a variety of in vitro transcription kits commercially available.

According to the invention, the stability and translation efficiency of the RNA can be modified as necessary. For example, RNA can be stabilized and its translation increased by one or more modifications that have stabilizing effects and/or increase the efficiency of RNA translation. To increase the expression of the RNA used in accordance with the present invention, it can be modified within the coding region, that is, the sequence encoding the expressed peptide or protein, preferably without altering the sequence of the expressed peptide or protein, to increase the GC content to increase mRNA stability and perform codon optimization and thus improve translation in cells.

The term “modification” in the context of the RNA used in the present invention includes any modification of an RNA that is not naturally present in said RNA.

In one embodiment of the invention, the RNA used according to the invention does not have uncapped 5'-triphosphates. Removal of such uncapped 5'-triphosphates can be achieved by treating the RNA with a phosphatase.

The RNA according to the invention may have modified ribonucleotides to increase its stability and/or decrease its cytotoxicity. For example, in one embodiment, in the RNA used according to the invention, cytidine is partially or completely replaced, preferably completely, by 5-methylcytidine. Alternatively or additionally, in one embodiment, in the RNA used according to the invention, uridine is partially or completely, preferably completely, replaced by pseudouridine.

In one embodiment, the term "modification" refers to providing an RNA with a 5' cap or a 5' cap analogue. The term “5' cap” refers to a cap structure found at the 5' end of an mRNA molecule and generally consists of a guanosine nucleotide connected to the mRNA by an unusual 5' to 5' triphosphate bond. In one embodiment, this guanosine is methylated at the 7-position. The term “conventional 5' cap” refers to a naturally occurring RNA 5' cap, preferably the 7-methylguanosine (m7G) cap. In the context of the present invention, the term “5' cap” includes a 5' cap analog that resembles the structure of the RNA cap and is modified to possess the ability to stabilize RNA and/or enhance translation. of the RNA if it is bound to it preferably in vivo and/or in a cell.

The RNA may comprise additional modifications. For example, a further modification of the RNA used in the present invention may be an extension or truncation of the natural poly(A) tail or an alteration of the 5' or 3' untranslated regions (UTR), such as the introduction of a UTR that is not related to the coding region of said RNA, for example, the exchange of the existing 3'-UTR with or the insertion of one or more, preferably two copies of a 3'-UTR derived from a globin gene, such as alpha2-globin, alpha1-globin, beta-globin, preferably beta-globin, more preferably human beta-globin.

RNA that has an unmasked poly-A sequence is translated more efficiently than RNA that has a masked poly-A sequence. The term “poly(A) tail” or “poly-A sequence” refers to a sequence of adenyl (A) residues typically found at the 3' end of an RNA molecule and “unmasked poly-A sequence.” means that the poly-A sequence at the 3' end of an RNA molecule ends with an A of the poly-A sequence and is not followed by nucleotides other than A located at the 3' end, that is, downstream, of the poly-A sequence. Furthermore, a long poly-A sequence of approximately 120 base pairs results in optimal transcription stability and translation efficiency of the RNA.

Therefore, to increase the stability and/or expression of the RNA used according to the present invention, it can be modified so that it is present together with a poly-A sequence, preferably having a length of 10 to 500, more preferably 30 to 300, even more preferably 65 to 200 and especially 100 to 150 adenosine residues. In an especially preferred embodiment, the poly-A sequence has a length of approximately 120 adenosine residues. To further increase the stability and/or expression of the RNA used according to the invention, the poly-A sequence can be unmasked.

The term “stability” of RNA relates to the “half-life” of RNA. The “half-life” refers to the period of time it takes to eliminate half the activity, quantity or number of molecules. In the context of the present invention, the half-life of an RNA is indicative of the stability of said RNA. The half-life of RNA can influence the “duration of expression” of the RNA. RNA that has a long half-life can be expected to be expressed over a long period of time.

Of course, if according to the present invention it is desired to decrease the stability and/or efficiency of translation of the RNA, it is possible to modify the RNA to interfere with the function of the elements as described above by increasing the stability and/or efficiency of RNA translation.

“Encoding” refers to the inherent property of specific nucleotide sequences in a nucleic acid to serve as templates for the synthesis of other polymers and macromolecules in biological processes that have a defined sequence of nucleotides or a defined sequence of amino acids. Therefore, a nucleic acid encodes a protein if the expression (translation and optionally transcription) of the nucleic acid produces the protein in a cell or other biological system.

The term "expression" is used according to the invention in its most general meaning and comprises the production of RNA and/or peptides or polypeptides, for example by transcription and/or translation. With respect to RNA, the term “expression” or “translation” refers in particular to the production of peptides or polypeptides. It also includes the partial expression of nucleic acids. Furthermore, the expression can be transient or stable.

In the context of the present invention, the term "transcription" refers to a process in which the genetic code of a DNA sequence is transcribed into RNA. The RNA can then be translated into protein. According to the present invention, the term "transcription" comprises "in vitro transcription", where the term "in vitro transcription" refers to a process in which RNA, in particular mRNA, is synthesized in vitro in a system cell-free, preferably using appropriate cell extracts. Preferably, cloning vectors are applied for the generation of transcripts. These cloning vectors are generally called transcription vectors and, in accordance with the present invention, are encompassed by the term "vector." In accordance with the present invention, the RNA used in the present invention is preferably in vitro transcribed RNA (IVT-RNA) and can be obtained by in vitro transcription of an appropriate DNA template. The promoter to control transcription can be any promoter of any RNA polymerase. Particular examples of RNA polymerases are the T7, T3 and SP6 RNA polymerases. Preferably, the in vitro transcription according to the invention is controlled by a T7 or SP6 promoter. A DNA template for in vitro transcription can be obtained by cloning a nucleic acid, in particular cDNA, and introducing it into a vector appropriate for in vitro transcription. cDNA can be obtained by reverse transcription of RNA.

The term "translation" according to the invention refers to the process in the ribosomes of a cell by which a strand of messenger RNA directs the assembly of an amino acid sequence to form a peptide or polypeptide.

Expression control sequences or regulatory sequences, which according to the invention may be functionally linked to a nucleic acid, may be homologous or heterologous with respect to the nucleic acid. A coding sequence and a regulatory sequence are “functionally” linked to each other if they are covalently linked, such that transcription or translation of the coding sequence is under the control or influence of the regulatory sequence. If the coding sequence is to be translated into a functional protein, with functional linkage of a regulatory sequence to the coding sequence, induction of the regulatory sequence leads to a transcription of the coding sequence, without causing a frameshift in the coding sequence. or inability of the coding sequence to be translated into the desired protein or peptide.

The term “expression control sequence” or “regulatory sequence” comprises, according to the invention, promoters, ribosome binding sequences and other control elements that control the transcription of a nucleic acid or the translation of the derived RNA. In certain embodiments of the invention, the regulatory sequences can be controlled. The precise structure of the regulatory sequences may vary depending on the species or cell type, but generally comprises 5' untranscribed and 5' and 3' untranslated sequences, which are involved in the initiation of transcription or translation, such as Box TATA, limitation sequence, CAAT sequence and the like. In particular, the 5' non-transcribed regulatory sequences comprise a promoter region that includes a promoter sequence for transcriptional control of the functionally linked gene. Regulatory sequences may also comprise enhancer sequences or upstream activator sequences.

According to the invention, it is preferred that a nucleic acid such as RNA encoding a peptide or protein once absorbed or introduced, i.e. transfected or transduced, into a cell which cell may be present in vitro or in a subject results in the expression of said peptide or protein. The cell may express the encoded peptide or protein intracellularly (e.g., in the cytoplasm and/or in the nucleus), may secrete the encoded peptide or protein, or may express it on the surface.

In accordance with the invention, terms such as "expressing nucleic acid" and "nucleic acid encoding" or similar terms are used interchangeably herein and with respect to a particular peptide or polypeptide mean that the nucleic acid, if present in the appropriate environment, preferably within a cell, it can be expressed to produce said peptide or polypeptide.

According to the invention, any means can be used to engineer cells in order to increase the cellular level of CFLAR. In one embodiment, cells are engineered to increase the cellular level of CFLAR by transfecting the cells with nucleic acid encoding a CFLAR polypeptide.

Terms such as “transfer”, “introduce”, “transfect” or “transduce” are used interchangeably herein and relate to the introduction of nucleic acids, in particular exogenous or heterologous nucleic acids, such as RNA into a cell. According to the present invention, the cell may be present in vitro or in vivo, for example, the cell may be part of an organ, a tissue and/or an organism. According to the invention, the transfection can be transient or stable. For some transfection applications, it is sufficient that the transfected genetic material is expressed only transiently. Since the nucleic acid introduced in the transfection process is normally not integrated into the nuclear genome, the foreign nucleic acid will be diluted by mitosis or degraded. Cell lines that allow episomal amplification of nucleic acids greatly reduce the dilution rate. If the transfected nucleic acid is to actually remain in the genome of the cell and its daughter cells, stable transfection must occur. RNA can be transfected into cells to transiently express its encoded protein.

The term “cellular FLICE inhibitory protein [IL-1p converting enzyme-like Fas-associated death domain (FADD)]”, “cellular FLICE inhibitory protein”, “CFLAR” or “c-FLIP” is an antiapoptotic master regulator. and resistance factor that suppresses apoptosis induced by tumor necrosis factor a (TNF-a), Fas-L and TNF-related apoptosis-inducing ligand (TRAIL), as well as apoptosis triggered by chemotherapy agents in malignant cells . CFLAR is expressed as (CFLAR<l>) long, (CFLAR) short and CFLAR<r>splice variants in human cells. CFLAR binds to FADD and/or caspase-8 or -10 and TRAIL receptor 5 (DR5) in a ligand-dependent and -independent manner and forms an apoptosis inhibitory complex (AIC). This interaction, in turn, prevents the formation of the death-inducing signaling complex (DISC) and subsequent activation of the caspase cascade. CFLAR<l>and CFLARs are also known to have multifunctional functions in various signaling pathways, in addition to activating and/or positively regulating several cytoprotective and survival signaling proteins, including Akt, ERK and NF-κB. According to the invention, the term "CFLAR" includes any variants, in particular mutants, splice variants, conformations, isoforms, allelic variants, species variants and species homologs, in particular those that are naturally present, and comprises CFLAR<l>, CFLARs, CFLAR<r>and v-CFLAR.

The term “CFLAR<l>” preferably refers to a protein comprising the amino acid sequence according to SEQ ID NO: 1 of the sequence listing or a variant of said amino acid sequence. In one embodiment, the amino acid sequence according to SEQ ID NO: 1 of the sequence listing is encoded by the nucleic acid sequence according to SEQ ID NO: 2 of the sequence listing.

The term "CFLARs" preferably refers to a protein comprising the amino acid sequence according to SEQ ID NO: 3 of the sequence listing or a variant of said amino acid sequence. In one embodiment, the amino acid sequence according to SEQ ID NO: 3 of the sequence listing is encoded by the nucleic acid sequence according to SEQ ID NO: 4 of the sequence listing.

The term “CFLAR<r>” preferably refers to a protein comprising the amino acid sequence according to SEQ ID NO: 5 of the sequence listing or a variant of said amino acid sequence. In one embodiment, the amino acid sequence according to SEQ ID NO: 5 of the sequence listing is encoded by the nucleic acid sequence according to SEQ ID NO: 6 of the sequence listing.

The term "v-CFLAR" preferably refers to a protein comprising the amino acid sequence according to SEQ ID NO: 7 of the sequence listing or a variant of said amino acid sequence. In one embodiment, the amino acid sequence according to SEQ ID NO: 7 of the sequence listing is encoded by the nucleic acid sequence according to SEQ ID NO: 8 of the sequence listing.

According to the present invention, the term "peptide" refers to substances comprising two or more, preferably 3 or more, preferably 4 or more, preferably 6 or more, preferably 8 or more, preferably 10 or more, preferably 13 or more, preferably 16 more, preferably 21 or more and up to preferably 8, 10, 20, 30, 40 or 50, in particular 100 amino acids covalently linked by peptide bonds.

The term "protein" refers to large peptides, that is, polypeptides, preferably peptides with more than 100 amino acid residues, but in general the terms "peptide", "polypeptide" and "protein" are synonyms and are used interchangeably in This document.

The present invention also includes "variants" of the peptides, proteins or amino acid sequences described herein.

For the purposes of the present invention, "variants" of an amino acid sequence comprise amino acid insertion variants, amino acid addition variants, amino acid deletion variants and/or amino acid substitution variants.

Amino acid insertion variants comprise insertions of one or two or more amino acids in a particular amino acid sequence. In the case of amino acid sequence variants that have an insertion, one or more amino acid residues are inserted at a particular site in an amino acid sequence, although a random insertion is also possible with appropriate screening of the resulting product.

Amino acid addition variants comprise amino and/or carboxy terminal fusions of one or more amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids.

Amino acid deletion variants are characterized by the deletion of one or more amino acids from the sequence, such as by the deletion of 1,2, 3, 5, 10, 20, 30, 50 or more amino acids. Deletions can be at any position on the protein. Amino acid deletion variants that comprise deletion at the N-terminal and/or C-terminal end of the protein are also called N-terminal and/or C-terminal truncation variants.

Amino acid substitution variants are characterized by the deletion of at least one residue from the sequence and the insertion of another residue in its place. Modifications in positions of the amino acid sequence that are not conserved between homologous proteins or peptides and/or the substitution of amino acids with others that have similar properties are preferred. Preferably, the amino acid changes in protein variants are conservative amino acid changes, that is, substitutions of similarly charged or uncharged amino acids. A conservative amino acid change involves the substitution of one of a family of amino acids that are related in their side chains. Natural amino acids are generally divided into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged. Polar amino acids (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine are sometimes classified together as aromatic amino acids.

Preferably, the degree of similarity, preferably identity, between a given amino acid sequence and an amino acid sequence that is a variant of said given amino acid sequence will be at least about 60%, 65%, 70%, 80%, 81%. 82%, 83%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98% or 99%. The degree of similarity or identity is preferably provided for a region of amino acids that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%. %, at least about 70%, at least about 80%, at least about 90% or about 100% of the total length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is preferably provided for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180 or about 200 amino acids, preferably continuous amino acids. The degree of similarity or identity is preferably indicated for a segment of at least 80, at least 100, at least 120, at least 150, at least 180, at least 200 or at least 250 amino acids. In preferred embodiments, the degree of similarity or identity is provided for the entire length of the reference amino acid sequence. Alignment to determine sequence similarity, preferably sequence identity, can be performed with tools known in the art, preferably using the best sequence alignment, for example using Align, using standard configurations, preferably EMBOSS::needle, Matrix : Blosum62, Gap Open 10.0, Gap Extend 0.5.

“Sequence similarity” indicates the percentage of amino acids that are identical or that represent conservative amino acid substitutions. “Sequence identity” between two amino acid sequences indicates the percentage of amino acids that are identical between the sequences.

The term “percentage identity” is intended to designate a percentage of identical amino acid residues between the two sequences to be compared, obtained after the best alignment, this percentage being purely statistical and the differences between the two sequences being distributed randomly and throughout their length. Sequence comparisons between two amino acid sequences are conventionally carried out by comparing these sequences after they have been optimally aligned, such comparison being performed by segment or by "comparison window" to identify and compare local regions of sequence similarity. The optimal alignment of the sequences to be compared can be produced, in addition to manually, using the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, using the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, using the similarity search method of Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA85, 2444, or through computer programs that use these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

The percentage of identity is calculated by determining the number of identical positions between the two sequences being compared, dividing this number by the number of positions compared and multiplying the result obtained by 100 to obtain the percentage of identity between these two sequences.

The homologous amino acid sequences according to the invention have at least 40%, in particular at least 50%, at least 60%, at least 70%, at least 80%, at least 90% and preferably at least 95%, at least 98% or at least 99% identity of the amino acid residues.

The amino acid sequence variants described herein can be easily prepared by one skilled in the art, for example, by manipulation of recombinant DNA. The manipulation of DNA sequences to prepare peptides or proteins that have substitutions, additions, insertions or deletions is described in detail in Sambrook et al. (1989), for example. Furthermore, the peptides and amino acid variants described herein can be easily prepared with the aid of known peptide synthesis techniques such as, for example, by solid phase synthesis and similar methods.

The invention includes derivatives of the peptides or proteins described herein that are encompassed by the terms "peptide" and "protein." According to the invention, protein and peptide “derivatives” are modified forms of proteins and peptides. Such modifications include any chemical modification and comprise single or multiple substitutions, deletions and/or additions of any molecule associated with the protein or peptide, such as carbohydrates, lipids and/or proteins or peptides. In one embodiment, protein or peptide “derivatives” include those modified analogues that result from glycosylation, acetylation, phosphorylation, amidation, palmitoylation, myristoylation, isoprenylation, lipidation, alkylation, derivatization, introduction of protecting/blocking groups, proteolytic cleavage or ligation. to an antibody or another cellular ligand. The term "derivative" also extends to all functional chemical equivalents of said proteins and peptides. Preferably, a modified peptide has greater stability and/or greater immunogenicity.

According to the invention, a variant or derivative of a peptide or protein preferably has a functional property of the peptide or protein from which it is derived. Preferably, a variant or derivative of a CFLAR protein has the same or similar property as the CFLAR protein from which it is derived.

The term "derived" means according to the invention that a particular entity, in particular a particular sequence, is present in the object from which it is derived, in particular an organism or molecule. In the case of amino acid sequences, especially particular sequence regions, “derived” means in particular that the relevant amino acid sequence is derived from an amino acid sequence in which it is present.

The term "cell" or "host cell" is preferably an intact cell, that is, a cell with an intact membrane that has not released its normal intracellular components such as enzymes, organelles or genetic material. An intact cell is preferably a viable cell, that is, a living cell capable of carrying out its normal metabolic functions. Preferably, said term refers according to the invention to any cell that can be transformed or transfected with an exogenous nucleic acid. The term "cell" includes according to the invention prokaryotic cells (for example, E. coli) or eukaryotic cells (for example, dendritic cells, B cells, CHO cells, COS cells, K562 cells, HEK293 cells, HELA cells, cells yeast and insect cells). The exogenous nucleic acid can be found within the cell (i) freely dispersed as such, (ii) incorporated into a recombinant vector, or (iii) integrated into the host cell genome or mitochondrial DNA. Mammalian cells are especially preferred, such as cells from humans, mice, hamsters, pigs, goats and primates. Cells can be derived from a large number of tissue types and include primary cells and cell lines.

A cell comprising a nucleic acid, for example that has been transfected with a nucleic acid, preferably expresses the peptide or protein encoded by the nucleic acid.

The term “expansion” refers to a process in which a specific entity multiplies. In one embodiment of the present invention, the term is used in the context of an immunological response in which lymphocytes are stimulated by an antigen, proliferate and the specific lymphocyte that recognizes said antigen is amplified. Preferably, clonal expansion leads to lymphocyte differentiation.

The tolerogenic or immunosuppressive cells described herein (either generated in vitro for administration to a subject or generated in situ in a subject, for example, by administration of a CFLAR-encoding nucleic acid) are useful in suppression methods. of an immune response in a mammal for the treatment or prevention of an immune disease or an inflammatory disease, or rejection of transplants that include organs, tissues, cell and/or protein transplantation. Without wishing to be bound by any particular theory, tolerogenic or immunosuppressive cells that are administered to the patient or formed in place in the patient inhibit the activation and proliferation of the patient's T cells.

Tolerogenic or immunosuppressive cells are useful for preventing or suppressing side effects resulting from the patient's immune response mounted against the protein administered to the patient (optionally by expressing a nucleic acid administered to a patient), thereby decreasing the protein efficacy and safety. Protein therapies include, but are not limited to, monoclonal antibodies, enzymes, cytokines, and toxins.

Furthermore, tolerogenic or immunosuppressive cells are useful for reducing and/or eliminating an immune response to a transplant in a recipient by administering or generating in the transplant recipient a number of tolerogenic or immunosuppressive cells effective to reduce or inhibit host rejection of the transplant. . The transplant may include a biocompatible network or a tissue, organ, cell or molecule from a donor to be transplanted. An example of a transplant may include, but is not limited to, skin cells or tissue, bone marrow, and solid organs such as heart, pancreas, kidney, lung, and liver. In some cases, the transplant is a nucleic acid or protein. According to the disclosure provided herein, myeloid cells or their progenitor cells can be obtained from any source, for example, from the transplant donor, the transplant recipient, or from another unrelated source (a completely different individual or species). Tolerogenic or immunosuppressive cells are preferably administered or generated in the recipient before or simultaneously with a transplant to reduce and/or eliminate rejection of the transplant by the host. In yet another embodiment, tolerogenic or immunosuppressive cells may be administered or generated in the transplant recipient after administration of the transplant.

The present disclosure relates to the use of tolerogenic or immunosuppressive cells as therapy to inhibit graft-versus-host disease after transplantation. In this disclosure, the present invention encompasses a method of contacting a donor transplant with tolerogenic or immunosuppressive cells prior to transplantation to a recipient. Tolerogenic or immunosuppressive cells serve to enhance, inhibit or reduce an adverse response of the transplanted donor against the recipient. Tolerogenic or immunosuppressive cells can be obtained from any source, for example, from the transplant donor, the transplant recipient, or from another unrelated source (a completely different individual or species) for the use of eliminating or reducing an unwanted immune response. through a transplant. against a transplant recipient. Accordingly, the tolerogenic or immunosuppressive cells may be autologous, allogeneic, or xenogeneic to the transplant donor, transplant recipient, or an otherwise unrelated source. Preferably, a transplant recipient suffering from graft-versus-host disease or at risk of graft-versus-host disease can be treated by administering the tolerogenic or immunosuppressive cells to the recipient or by generating the tolerogenic or immunosuppressive cells in the recipient to reduce, inhibit or eliminate the severity of graft versus host disease. Preferably, the tolerogenic or immunosuppressive cells can be obtained from the recipient before transplantation and can be stored and/or expanded in culture to provide a reserve of tolerogenic or immunosuppressive cells in quantities sufficient to treat an ongoing graft versus host reaction. However, as discussed elsewhere herein, tolerogenic or immunosuppressive cells can be obtained from any source, for example, from the transplant donor, the transplant recipient, or from an unrelated source (a completely different individual or species). ).

Based on the disclosure herein, it is anticipated that the tolerogenic or immunosuppressive cells described herein may be used in conjunction with current modes of treatment, for example the use of immunosuppressive drug therapy for the treatment of host rejection. to donor tissue or graft-versus-host disease. An advantage of using tolerogenic or immunosuppressive cells together with immunosuppressive drugs in transplants is that by using tolerogenic or immunosuppressive cells to improve the severity of the immune response in a transplant recipient, the amount of immunosuppressive drug therapy used and/or the frequency of Administration of immunosuppressive drug therapy may be reduced. One benefit of reducing the use of immunosuppressive drug therapy is the relief of overall immune suppression and unwanted side effects associated with immunosuppressive drug therapy.

Described herein, in particular, is a method of preventing or treating transplant rejection and/or graft-versus-host disease by administering to a patient or generating in a patient tolerogenic or immunosuppressive cells described herein in a prophylactically or therapeutically effective amount for the prevention, treatment or amelioration of transplant host rejection and/or graft versus host disease. According to the present disclosure, a therapeutically effective amount of tolerogenic or immunosuppressive cells is an amount that inhibits or decreases the number of activated T cells, compared to the number of activated T cells in the absence of the administration of tolerogenic or immunosuppressive cells. In the situation of transplant rejection by the host, an effective amount of tolerogenic or immunosuppressive cells is an amount that inhibits or decreases the number of activated T cells in the transplant recipient compared to the number of activated T cells in the recipient. before the administration of tolerogenic or immunosuppressive cells. In the case of graft versus host disease, an effective amount of tolerogenic or immunosuppressive cells is an amount that inhibits or decreases the number of activated T cells present in the transplant.

An effective number of tolerogenic or immunosuppressive cells can be determined by comparing the number of activated T cells in a recipient or transplant prior to administration of tolerogenic or immunosuppressive cells thereto, with the number of activated T cells present in the recipient or transplant. . after the administration of tolerogenic or immunosuppressive cells to it. A decrease, or lack of increase, in the number of activated T cells in the transplant recipient or in the transplant itself that is associated with the administration of tolerogenic or immunosuppressive cells thereto, indicates that the number of tolerogenic or immunosuppressive cells is reduced. administers a therapeutically effective amount of immunosuppressive or immunosuppressive tolerogenic cells.

Instead of administering tolerogenic or immunosuppressive cells to a patient, the invention can also be used to express CFLAR in cells of a mammal to generate tolerogenic or immunosuppressive cells in the mammal. This aspect of the invention relates to a type of gene therapy in which therapeutic proteins are administered to a mammal by introducing a nucleic acid that encodes them into cells of a mammal. In all cases in which a nucleic acid is transfected into a cell, the nucleic acid sequence encoding CFLAR is operably linked to regulatory sequences necessary to achieve expression of the nucleic acid sequence in the cell. The nucleic acid construct is preferably provided as an expression vector that includes the coding sequence for CFLAR operatively linked to essential regulatory sequences so that when the vector is transfected into the cell, the cell will express the coding sequence. The coding sequence is operatively linked to the regulatory elements necessary for the expression of that sequence in cells. The nucleic acid encoding CFLAR may be cDNA, genomic DNA, synthesized DNA or a hybrid thereof or an RNA molecule such as mRNA.

The nucleic acid construct includes the nucleotide sequence encoding CFLAR operatively linked to the regulatory elements and may remain present in the cell as a functional cytoplasmic molecule, a functional episomal molecule, or may be integrated into the chromosomal DNA of the cell. Exogenous genetic material can be introduced into cells where it remains as separate genetic material in the form of a plasmid.

Alternatively, linear DNA that can integrate into the chromosome can be introduced into the cell. By introducing DNA into the cell, reagents can be added that promote the integration of the DNA into the chromosomes. DNA sequences that are useful in promoting integration may also be included in the DNA molecule. Alternatively, RNA can be introduced into the cell.

The basic approach of using tolerogenic or immunosuppressive cells of the invention to induce tolerance has broad application as an adjunct to therapies that use a potentially immunogenic molecule for therapeutic purposes. For example, an increasing number of therapeutic approaches utilize a protein molecule, such as an antibody, a fusion protein, or the like, for the treatment of a clinical disorder. A limitation to the therapeutic use of such molecules is that they may provoke an immune response directed against the therapeutic molecule in the subject being treated (for example, the efficacy of Factor VIII in human subjects is hindered by the induction of an immune response against Factor VIII in the human subject). The present invention is an improvement of conventional protein therapy in the context of inducing tolerance against the administered molecule.

Administration of tolerogenic or immunosuppressive cells of the invention to inhibit T cell responses can be applied to these therapeutic situations to allow long-term use of the therapeutic molecule in the subject without eliciting an immune response.

According to the invention, the term "disease" or "disorder" refers to any pathological state.

The term “inflammatory disease” refers to any disease that is characterized or associated with high levels of inflammation in tissues, particularly connective tissues, or degeneration of these tissues. A chronic inflammatory disease is a medical condition characterized by persistent inflammation. Examples of inflammatory (chronic) diseases include celiac disease, vasculitis, lupus, chronic obstructive pulmonary disease (COPD), irritable bowel disease, atherosclerosis, arthritis, ankylosing spondylitis, Crohn's disease, colitis, chronic active hepatitis, dermatitis, and psoriasis.

The term “immune disease” or “autoimmune disorder” refers to any disease/disorder in which the body produces an immunogenic (i.e., immune system) response to some constituent of its own tissue. In other words, the immune system loses its ability to recognize any tissue or system within the body as its own and attacks it as if it were foreign. Autoimmune diseases can be classified into those in which predominantly one organ is affected (e.g., hemolytic anemia and autoimmune thyroiditis) and those in which the immune disease process spreads through many tissues (e.g., lupus erythematosus). systemic). For example, multiple sclerosis is thought to be caused by T cells attacking the sheaths surrounding nerve fibers in the brain and spinal cord. This results in loss of coordination, weakness and blurred vision. Examples of autoimmune diseases include, but are not limited to, Addision's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, diabetes (Type I), dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Dystrophic epidermolysis bullosa, Guillain-Barr disease, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema , pernicious anemia, ulcerative colitis and type I diabetes mellitus, among others.

“Transplant” refers to a biocompatible network or a tissue, organ or cell from a donor, to be transplanted. An example of a transplant may include, but is not limited to, skin cells or tissue, bone marrow, and solid organs such as heart, pancreas, kidney, lung, and liver. A transplant can also refer to any material that is to be administered to a host. For example, a transplant can refer to a nucleic acid or a protein.

The term “transplant rejection” refers to the rejection of a transplant, such as a transplanted tissue or organ, by the recipient's immune system, which may ultimately destroy the transplant.

The term “graft versus host disease” or “GvHD,” as used herein, is defined as a condition in a mammal, including humans, that occurs when immunologically allogeneic cells are introduced, transplanted, or engrafted. active, such as those of the bone marrow or spleen, in a host that produces a severe, even fatal, reaction due to immunoincompatibility between the host and the graft in which the immunocompetent cells of the graft immunologically attack the host.

By "treat" is meant administering an agent or composition as described herein to a subject for the purpose of preventing or eliminating a disease, including stopping or slowing a disease in a subject; inhibit or delay the development of a new disease in a subject; reduce the frequency or severity of symptoms and/or recurrences in a subject who currently has or has previously had a disease; and/or prolong, that is, increase the useful life of the subject. In particular, the term “treatment of a disease” includes curing, shortening the duration, ameliorating, preventing, slowing or inhibiting the progression or worsening of, or preventing or delaying the onset of, a disease or the symptoms thereof.

By “being at risk” we mean a subject, that is, a patient, who is identified as having a higher than normal probability of developing a disease compared to the general population. Furthermore, a subject who has had, or currently has, a disease is a subject who has an increased risk of developing a disease, as such subject may continue to develop a disease. Furthermore, a subject who has received or will receive a transplant is a subject who has an increased risk of developing GvHD.

In the context of the present invention, terms such as "protect", "prevent", "prophylactic", "preventive" or "protective" relate to the prevention or treatment or both of the onset and/or spread of a disease. in a subject and, in particular, to minimize the possibility of a subject developing a disease or to delay the development of a disease. For example, a person at risk for a disease would be a candidate for therapy to prevent a disease.

A prophylactic administration of an agent or composition described herein preferably protects the recipient from the development of a disease. A therapeutic administration of an agent or composition described herein may lead to inhibition of disease progression/growth. This comprises slowing the progress/growth of the disease, in particular an interruption of the progress of the disease, preferably leading to elimination of the disease.

The agents and compositions provided herein can be used alone or in combination with conventional therapeutic regimens such as surgery, irradiation, chemotherapy and/or bone marrow transplantation (autologous, syngeneic, allogeneic or unrelated).

The term “live” is related to the situation of a subject.

The terms “subject”, “individual”, “organism” or “patient” are used interchangeably and relate to vertebrates, preferably mammals. For example, mammals in the context of the present invention are humans, non-human primates, domestic animals such as dogs, cats, sheep, cows, goats, pigs, horses, etc., laboratory animals such as mice, rats, rabbits. , guinea pigs, pigs, etc. as well as animals in captivity such as zoo animals. The term “animal” as used herein also includes humans. The term "subject" may also include a patient, that is, an animal, preferably a human being that has a disease, preferably a disease as described herein.

The term “autologous” is used to describe anything that is derived from the same subject. For example, “autologous transplant” refers to a transplant of tissue or organs derived from the same subject. Such procedures are advantageous because they overcome the immunological barrier that would otherwise lead to rejection.

The term “allogeneic” is used to describe anything that is derived from different individuals of the same species. Two or more individuals are said to be allogeneic to each other when the genes at one or more loci are not identical.

The term “sygeneic” is used to describe anything that is derived from individuals or tissues that have identical genotypes, that is, identical twins or animals of the same inbred strain, or their tissues.

The term “heterologous” is used to describe something that consists of multiple different elements. For example, the transfer of bone marrow from one individual to another individual constitutes a heterologous transplant. A heterologous gene is a gene derived from a source other than the subject.

The agents and compositions described herein may be administered by any conventional route, including by injection or infusion. Administration can be carried out, for example, orally, intravenously, intraperitoneally, intramuscularly, subcutaneously or transdermally. In particular, the cells described herein may be administered systemically, that is, parenterally, by intravenous injection or may be directed to a particular tissue or organ, such as the bone marrow. Additionally, the cells can be administered by subcutaneous implantation of cells or by injection of cells into connective tissue, for example, muscle.

The agents and compositions described herein are administered in effective amounts. An “effective amount” refers to the amount that achieves a desired reaction or desired effect, for example, a detectable level of immunosuppression or tolerance, alone or in conjunction with additional doses. In the case of the treatment of a particular disease or a particular condition, the desired reaction preferably relates to the inhibition of the course of the disease. This includes slowing the progress of the disease and, in particular, interrupting or reversing the progress of the disease. The desired reaction in a treatment of a disease or condition may also be a delay in the onset or a prevention of the onset of said disease or condition.

An effective amount of an agent or composition described herein will depend on the condition to be treated, the severity of the disease, the individual parameters of the patient, including age, physiological condition, size and weight, duration of treatment , the type of accompaniment therapy (if present), the specific route of administration and similar factors. Accordingly, the administered doses of the agents and compositions described herein may depend on several of said parameters. In the event that a reaction in a patient is insufficient with an initial dose, higher doses (or effectively higher doses achieved by a different, more localized route of administration) may be used.

The pharmaceutical compositions described herein are preferably sterile and contain an effective amount of the therapeutically active substance to generate the desired reaction or desired effect.

The pharmaceutical compositions described herein are generally administered in pharmaceutically compatible amounts and in pharmaceutically compatible preparations. The term "pharmaceutically compatible" refers to a non-toxic material that does not interact with the action of the active component of the pharmaceutical composition. Preparations of this type may normally contain salts, buffer substances, preservatives, carriers and, where appropriate, other therapeutically active compounds. When used in medicine, salts must be pharmaceutically compatible. However, salts that are not pharmaceutically compatible can be used to prepare pharmaceutically compatible salts and are included in the invention. Pharmacologically and pharmaceutically compatible salts of this type include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic and similar acids. Pharmaceutically compatible salts can also be prepared as alkali metal salts or alkaline earth metal salts, such as sodium salts, potassium salts or calcium salts.

The pharmaceutical compositions described herein may comprise a pharmaceutically compatible carrier. The term “carrier” refers to an organic or inorganic component, natural or synthetic in nature, in which the active component is combined to facilitate application. According to the invention, the term "pharmaceutically compatible carrier" includes one or more compatible solid or liquid fillers, diluents or encapsulating substances, which are suitable for administration to a patient. The components of the pharmaceutical compositions described herein are generally such that no interaction occurs that substantially impairs the desired pharmaceutical efficacy.

The pharmaceutical compositions described herein may contain suitable buffer substances such as acetic acid in a salt, citric acid in a salt, boric acid in a salt and phosphoric acid in a salt.

The pharmaceutical compositions may also contain, if appropriate, suitable preservatives such as benzalkonium chloride, chlorobutanol, paraben and thimerosal.

The pharmaceutical compositions are usually provided in a uniform dosage form and may be prepared in a manner known per se. The pharmaceutical compositions described herein may be in the form of capsules, tablets, lozenges, solutions, suspensions, syrups, elixirs or in the form of an emulsion, for example.

Compositions suitable for parenteral administration typically comprise a sterile aqueous or non-aqueous preparation of the active compound, which is preferably isotonic to the blood of the recipient. Examples of compatible vehicles and solvents are Ringer's solution and isotonic sodium chloride solution. In addition, fixed oils, normally sterile, are used as solution or suspension medium.

The present invention is best illustrated by the following examples which should not be considered limiting the scope of the invention.

Examples

The techniques and methods used herein are described herein or are carried out in a manner known per se and as described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, second edition ( 1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. All methods, including the use of kits and reagents, are carried out according to the manufacturer's information, unless specifically indicated.

Example 1: Transduction of human stem cells (CD34+ cells) and monocytes (CD14+ cells) with specific lentiviruses encoding CFLAR and CFLARs promote an immunosuppressive phenotype.

We created two plasmid constructs containing the sequences of CFLAR<l>(Figure 1A) or CFLARs (Figure 1B). The two constructs contained a reporter gene: a green fluorescent protein (GFP) with tracking and marker function. For the production of recombinant lentiviral proteins, the calcium chloride precipitation method was used. Briefly, the plasmid vectors encoding key proteins for virus assembly were purchased by the Takara company (Takara Clontech, CA, USA). Calcium chloride CaCh (2 mM) to the solution containing the pGp vector, the pE-eco vector and the vector encoding CFLAR<l>.<o>CFLARs or luciferase (as control) (25 |jg). Subsequently, 2X HBS buffer (274 mM NaCh, 10 mM KCl, 1.5 mM Na2HPO4, 12 mM dextrose, 42 mM Hepes, pH 7.1) was added with stirring. The transfection mixture was then added dropwise to the 293T cell culture plate and the medium containing the viral particles was collected after 48 hours, centrifuged and filtered on 0.45 μm pore filters to remove the cells. and the remains and concentrated by ultracentrifugation at 50,000 g for 140 minutes at room temperature. Infection was carried out by spin inoculation: cells (human stem cells or monocytes) were placed in a swinging trough rotor of multi-well plates and placed in a centrifuge; The plate was spun at 2500 rpm for 4 hours at 30°C. After this step, we set up immunosuppressive assays. In the case of human stem cells (Bone Marrow CD34 cells, 1 million, cat. 2M-101C, Lonza, Switzerland), infected CD34 + cells were differentiated in vitro in the presence of G-CSF (40 ng/ml; Miltenyi Biotec Srl, Italy) and<g>M-CSF (40 ng/ml, Miltenyi Biotec Srl, Italy) for four days to promote the acquisition of m Ds C-related immunosuppressive activity. After four days, the cells recovered and were cocultured in the presence of PBMCs labeled with traces of activated cells (Figure 2A, left panel). Briefly, PBMCs were recovered and washed in IMDM medium (Lonza, Switzerland), supplemented with 10% FBS (EuroClone, Italy), 100 U/ml penicillin/streptomycin (Lonza, Switzerland), pmercaptoethanol (Sigma-Aldrich, Italy) and HEPES 10 mM (Lonza, Switzerland). PBMCs were resuspended to a final concentration of 107 cells/ml in PBS and stained with 2.5 μM as final working concentration of Cell Trace Violet stock solution (Invitrogen Molecular Probe, UK), followed by 5 min incubation at 37°. C, protected from light. The dye diffuses easily into cells, where intracellular esterases cleave it to produce a highly fluorescent compound. This compound covalently binds to intracellular amines, resulting in a stable and well-retained fluorescent dye. After each division, the dye is evenly distributed in the daughter cells, so cellular fluorescence decreases by half. In a flow cytometry histogram, discrete peaks appear toward the left side and represent each cell division or generation. The cells were then washed and resuspended in culture medium. Labeled “target” PBMCs were stimulated with 1 μg/ml coated anti-CD3 (clone, OKT-3; eBioscience, CA, USA) and 5 μg/ml soluble anti-CD28 (clone, CD28.2 , eBioscience, CA, USA) for four days and co-cultured with CD34+ “effector” cells differentiated in vitro into different effectors at target ratios in flat-bottom 384-well plates (BD Biosciences, New Jersey, USA). . Cell cultures were incubated at 37 °C and 5% CO in arginine- and glutamine-free RPMI (Biochrom AG, Germany), supplemented with 2 mM L-glutamine, 150 μM arginine, 10% FBS (Sigma-Aldrich, MO , USA), 10 U/ml penicillin and streptomycin (Lonza, Switzerland) and 0.1 mM HEPES (Lonza, Switzerland). At the end of culture, cells were stained with PE-Cy7-conjugated anti-CD3 (UCHT1, eBioscience, CA, USA) and all samples were acquired with a FACS-CantoII (BD Biosciences, NJ, USA). .) and a cell tracking signal from the selected lymphocytes were analyzed with FlowJo software (Treestar Inc.). The degree of cell proliferation was quantified by analyzing the number of proliferating cells, which is assumed to be 100% without CD34+ cells differentiated in vitro. We demonstrated that overexpression of CFLAR increased the immunosuppressive capacity to differentiate MDSC in vitro, suggesting a new biological role for this protein. Indeed, CFLAR-expressing CD34+ cells showed significantly increased suppressive activity compared to luciferase-expressing CD34+ cells. (Figure 2A, right panel). To further validate these data, we immunomagnetically infected (cat. 130-050-201; Miltenyi Biotec Srl, Italy) CD14+ monocytes purified from buffy coats, which generally do not show any immunosuppressive function, with lentiviruses expressing CFLAR or luciferase and cocultured them in the presence of PBMCs labeled with cellular traces activated by anti-human CD3/CD28 (Figure 2B, left panel). Also in this experimental setting, CFLAR-infected CD14+ cells acquired a stronger suppressive capacity compared to luciferase-infected monocytes (Figure 2B, right panel). This potent immunosuppressive function did not correlate with better survival of CFLAR-expressing CD14+ cells compared to controls, as the percentage of CD14+7-AAD'ANNEXIN-V' cells after in vitro co-culture was comparable (data not shown ) using flow cytometry using the APC-AnnexinV Apoptosis Detection Kit with 7-AAD (cat. 640930, Biolegend, CA, USA). All samples were acquired with a FACS-CantoII (BD Biosciences, New Jersey, USA) and analyzed with FlowJo software (Treestar Inc.).

Example 2: Forced expression of CFLAR in monocytes drives an MDSC-like molecular program that controls immunosuppressive function.

To better identify the CFLAR-activated molecular pathway underlying the immunosuppressive functional program in myeloid cells, we performed transcriptome analysis of CFLAR- and luciferase-infected CD14+ cells. isolated from four healthy donors. Briefly, total RNA was isolated using TRIzol reagent (Life technology, CA, USA) and RNA integrity was assessed using Agilent-2100-Bioanalyzer (Agilent Technologies, CA,<e>USA). RNA was further purified using the RNeasy MinElute Cleanup kit (Qiagen, Venlo, The Netherlands) and cDNA was synthesized and amplified from total purified RNA using Ovation Pico WTA System V2 (NuGEN, CA, USA). All samples were hybridized to Affymetrix U133 PLUS 2.0 arrays and scanned with an Affymetrix GCS 30007G scanner. The sample and genes are clustered using Pearson's correlation coefficient and average as distance and linkage metrics, respectively. As shown in Figure 3, supervised clustering (q.val<0.05, fold change >2) generated a list of 750 upregulated (Figure 4) and 1130 downregulated (data not shown) genes in CFLAR-infected monocytes. compared to controls. In particular, these differentially expressed genes were found to be significantly enriched in categories involved in inflammation pathways, Notch-related pathways, and IL-10-related pathway. Furthermore, forced expression of CFLAR triggers the upregulation of MDSC-related immunosuppressive genes, such as SOCS2, FAS, CCR7, CCL5, NF-kB, STAT3, CD38, CD274, CD273, IL4R, IL6, IL10, CFS3, PTGS2, and IDOl . Some of the assumed objectives related to CFLAR were tested and validated (Figure 5). In particular, we verified by real-time PCR that CFLAR-transduced monocytes showed upregulation of both CFLAR<l>and CFLARs, as well as upregulation of IDOl. To this end, we isolated total RNA from monocytes infected with CFLAR or luciferase using TRIzol reagent (Life technology, CA, USA) and RNA integrity was assessed using Agilent-2100-Bioanalyzer (Agilent Technologies, CA, USA). . RNA was further purified using the RNeasy MinElute Cleanup kit (Qiagen, Venlo, The Netherlands) and cDNA was synthesized and amplified from total purified RNA using Ovation Pico WTA System V2 (NuGEN, CA, USA). All samples were used to set up real-time PCR with custom primers designed by ABI Biosystems by Primer Express. Post-qRT-PCR analysis to quantify relative gene expression was performed using the comparative Ct (2-ññCt) method. As shown in Figure 5A, CFLAR-transduced monocytes expressed significantly high levels of putative target genes. Furthermore, we validated that CFLAR-transduced CD14+ cells secreted significantly higher levels of IL-10 than luciferase-transduced CD14+ cells by Enzyme Linked ImmunoSorbent Assay (ELISA) assay (R&D System, MN, USA). In short, this assay consists of transferring the culture medium into a specific 96-well plate previously incubated with purified antibody specific for IL-10. In some wells, instead of the sample, the calibration curve was plated using a 1:2 scalar dilution of the cytokine in question. After 2 h of incubation at 37°C, the plate was labeled with biotinylated anti-IL-10 antibody for 1 h, followed by 30 min of incubation at room temperature with peroxidase-linked streptavidin. The reaction of peroxidase with its substrate (TMB-3,3',5,5'-tetramethylbenzidine) allows the quantification of the cytokine of interest by reading at 450 nm. Finally, we verified that CFLAR expression imposed in monocytes promoted the upregulation of specific surface markers by flow cytometry. Briefly, CD14+ cells after 24 hours of infection, as described above, were resuspended in 100 μl of FACS buffer (0.9% NaCl solution containing 2% BSA and 0.02% NaN3; both from Sigma-Aldrich). with mouse anti-human D273-PE (cat. 557926, BD Biosciences, New Jersey, USA), mouse anti-human CD274-APC (cat. 228489, BD Biosciences, New Jersey, USA), Mouse anti-human CD80-APC (cat. 305220, Biolegend CA, USA), anti-human CD163-PE (cat.

556018, BD Biosciences, New Jersey, USA), anti-human CD44-PE (cat. 103024, Biolegend, New Jersey, USA), anti-human CD38-APC (cat. 554262, BD Biosciences, New Jersey, USA) . USA) and anti-human CD124-APC (cat. FAB23OP, R&D System, MN, USA). All samples were acquired with a FACS-CantoII (BD Biosciences, New Jersey, USA) and analyzed with FlowJo software (Treestar Inc.).

Example 3: Adoptive transfer of CFLAR-expressing monocytes controls the progression of GvHD in immunodeficient mice transplanted with human PBMC.

From these observations we decided to evaluate in vivo the immunosuppressive functions of CFLAR-infected CD14+ cells (as described above) to monitor the development of graft-versus-host disease (GvHD) using a xenogeneic mouse model. For this purpose, we intravenously (iv) injected sublethal irradiated (using 137Cs Source Irradiator), NOD.Cg-PrkdcscidIl2rgtm1Sug/JicTac (N<o>G) (purchased by Taconic, NY, USA) with 106 PBMC human cells isolated from buffy coats. When the frequency of circulating human CD3+ lymphocytes reached ~5% of the total cells, we began to treat the mice by intravenous transfer of 106 Monocytes that express CFLAR or luciferase. Briefly, through retro-orbital bleeding, we isolated 100 pl of blood; RBCs were removed by ACK (ammonium-potassium chloride) lysis buffer and cells were stained with rat anti-mouse CD45-APC (clone A20, Biolegend, NJ, USA), CD45-PerPC-Cy5 .5 anti-human (clone 2D1, Biolegend, NJ, USA) and anti-human CD3-PE-Cy7 (clone UHCT1, Biolegend, NJ, USA). All samples were acquired with a FACSCanto II (BD Biosciences, New Jersey, USA) and analyzed with FlowJo software (Treestar Inc.). Treatment was repeated four times, on days 21, 28, 42 and 49 after the initial challenge and the immunological, clinical and histopathological GvHD score of the mice was monitored (Figure 6A). Injection of CFLAR-infected CD14+ cells efficiently reduced the development of GvHD, resulting in better long-term survival of the transplanted mice compared to mice subjected to GvHD treated with luciferase-expressing monocytes or untreated (Figure 6B ). Furthermore, histological analysis and quantification of tissue-infiltrating human CD3+ T cells revealed less aggressive GvHD pathology in all analyzed organs of mice treated with CFLAR-expressing monocytes (Figure 6C). Briefly, tissues were removed necroscopically and fixed in 10% buffered formalin and embedded in paraffin. Histopathological evaluation was performed on standard hematoxylin and eosin sections. Pathological scoring was evaluated by two blinded pathologists using the previously published standard line guide (Cooke KR, Kobzik L, Martin TR, Brewer J, Delmonte J, Crawford JM, Ferrara JLM: An experimental model of idiopathic pneumonia syndrome after bone marrow transplantation : The roles of minor H antigens and endotoxin. Blood 8:3230, 199). Finally, paraffin sections of spleen, liver, lung, skin, colon, kidney and stomach were used for immunohistochemistry: the sections were stained with anti-human CD3 antibody (clone ab5690, Abcam, United Kingdom) to quantify infiltrating lymphocytes. in human tissues.

To test the ability of CFLAR-expressing monocytes to control the in vivo expansion of T cells in mice subjected to GvHD, we verified the presence and frequency of human CD3 + cells in mice treated four times on days 21, 28, 42 and 49 from of the PBMC graft (Figure 7A). After only two treatments, GvHD mice treated with CFLAR-expressing monocytes showed a lower frequency of human T cells compared to those treated with luciferase (Figure 7B). Notably, mice subjected to GvHD were sacrificed on day 35 after PBMC grafting and we assessed the frequency of human T cells by flow cytometry in the blood (Figure 7B i), spleen (Figure 7B ii), and bone marrow. (Figure 7B iii). For blood analysis, 100 μl of blood were isolated through retro-orbital bleeding; RBCs were removed using ACK (ammonium-chloride-potassium) lysing buffer and cells were stained with PECy7-conjugated anti-CD3 antibody (UCHT1, eBioscience, CA, USA). For spleen analysis, fresh splenocytes (106/sample) were resuspended in 100 μl of FACS buffer with anti-human FcyR blocking solution (cat. 130-046-702, Miltenyi Biotec S.r.l., Italy) for 10 minutes at room temperature to reduce nonspecific staining. After washing, cells were resuspended in FACS buffer and labeled with human PE-Cy7-conjugated anti-CD3 antibody (UCHT1, eBioscience, CA, USA). Finally, for bone marrow cell analysis, tibias and femurs were removed from mice undergoing GvHD using sterile techniques and the BM was washed. Red blood cells were removed by ACK (potassium ammonium chloride) lysis buffer and cells were stained with human PE-Cy7-conjugated anti-CD3 antibody (UCHT1, eBioscience, CA, USA). All samples were acquired with a FACSCanto II (BD Biosciences, New Jersey, USA) and analyzed with FlowJo software (Treestar Inc.). In addition, we also evaluated the activation status of human T cells. Briefly, splenocytes from GvHD-treated mice were stimulated or not (resting conditions) in vitro for 24 hours with phorbol 12-myristate 13-acetate (PMA, 50 ng/ml) and ionomycin (1 μg/ml). Splenocytes were washed and stained with surface antibodies (Pacific Blue-conjugated anti-human CD8 antibody (cat. 34417, Biolegend, CA, USA)), fixed, and permeabilized using the Perm/Wash kit (cat. 554723). , BD Biosciences, NJ, USA) and incubated with PE-labeled hlFN-Y antibody (cat. 506506, Biolegend, CA, USA) (Figure 7Biii). All samples were acquired with a FACSCanto II (BD Biosciences, New Jersey, USA) and analyzed with FlowJo software (Treestar Inc.). We demonstrated that T cells isolated from CFLAR-treated mice showed a statistically lower frequency of human CD8+ IFNy+ cells compared to those treated with luciferase, revealing a possible exhaustion or non-activated state mediated by CFLAR monocytes. Finally, we assessed that injection of CFLAR-expressing monocytes into mice subjected to GvHD induced the expansion of circulating regulatory T cells (Treg) compared to luciferase-expressing monocytes (Figure 7C).

Example 4: Rosa26.vFLIP;LysMcre mouse model

To better understand the molecular pathway induced by CFLAR, we took advantage of a transgenic mouse model that expresses the viral form of CFLAR: the Rosa26.vFLIP knock-in mice. Kaposi's sarcoma-associated herpesvirus encodes a FADD-like interferon-converting enzyme or caspase 8-inhibiting protein (FLICE) (<v>FLIP) in its genome. Rosa26.vFLIP knock-in mice were crossed with mice expressing Cre recombinase under the control of the endogenous liz2 protein (LysM-Cre), resulting in mice with vFLIP expression restricted to the myeloid cell lineage. Bone marrow cells and splenocytes were obtained from Rosa26.vFLIP mice; LysMcre and Rosa26.vFLIP and we isolated by fluorescence activated cell sorting (FACS) the CD11B.+ Ly6G' Ly6Calto and the CD11B+ Ly6G Ly6Clow subsets. The immunosuppressive activity of these cells was then evaluated by placing the freshly isolated myeloid cells in 96-well plates at a final concentration of 24% of the total cells in culture in the presence of splenocytes from 37B7 transgenic mice (a mouse model that presents all cells CD8+ T with a T cell receptor (TCR) designed specifically for the melanoma-associated antigen TRP2, labeled with 1 μM CFSE and diluted 1:10 with CD45.1+ splenocytes, in the presence of the TRP-2-180-188 peptide ( 1 μg/ml). T cells and the percentage of cells that had diluted CFSE were evaluated using FlowJo software (Treestar Inc.) The percentage of proliferating cells was then used to calculate the percentage of suppression of proliferation using the following formula: 1 - (% proliferation with myeloid cells / % proliferation without myeloid cells) * 100 (Figure 8).

Example 5: c-FLAR induces an immunosuppressive program independently of its antiapoptotic functions.

From buffy coats from healthy donors (HD), we purified and infected CD14+ monocytes with lentiviruses encoding c-FLAR or luciferase and demonstrated that the immune regulatory property of c-FLAR did not correlate with improved survival of monocytes expressing c -FLAR compared to controls, both when monocytes were cultured alone and when they were cultured with CD3/CD28-activated PBMCs. Indeed, the percentage of live (7-AAD'ANNEXIN-V') CD14+ cells at different time points are not significantly different between c-FLAR or luciferase monocytes (Figure 9a), indicating that immune regulatory activity is can separate from the antiapoptotic properties of c-FLAR, at least in the case of mature monocytes. Furthermore, to discriminate between pro-survival and induction of immunosuppressive function, we generated lentiviral expression vectors of other anti-apoptotic genes (i.e., Bcl-2 and Bcl-xL) and tested the immunosuppressive properties after their forced expression in monocytes. humans. As shown in Figure 9b, only forced expression of c-FLAR is able to significantly suppress in vitro T cell activation and proliferation.

Example 6: Forced expression of c-FLAR in monocytes drives MDSC-associated molecular programs.

Using gene ontology (GO) enrichment analysis, differentially expressed genes were significantly enriched in categories involved in inflammation, cytokine network, and genes upregulated in response to interferon proteins, as well as associated to the IL-10 pathway and, in general, to the NF-xB-related pathway (Figure 10a, b).

Example 7: c-FLAR activates the canonical NF-kB pathway in myeloid cells.

Forced expression of c-FLAR triggers the canonical NF-kB pathway. Gene expression data suggested unexpected transcriptional and signaling activity of c-FLAR, possibly related to the NF-<k>B pathway. To elucidate the molecular programs under the control of c-FLAR, we transiently transfected THP1 monocytic cells with in vitro transcribed (IVT) c-FLAR-encoding RNA. The rational thing was to induce sustained and virus-independent production of the target protein; Furthermore, IVT-RNA does not activate innate immune receptors or type I interferon production. The forced introduction of a synthetic c-FLAR RNA into THP1 cells triggered an immunosuppressive program consistent with the data shown so far with expression vectors of lentivirus (Figure 11a). c-FLAR RNA transfection significantly promoted the nuclear translocation of the p65 and p50 subunits of NF-kB, mediators of the canonical NF-kB activation pathway, while no difference was detected in the translocation of the p52 subunit (Figure 11b). To test whether NF-κB signaling was necessary for the post-FLAR immune regulatory program, we silenced the kinases IKKa (encoded by Chuk) and IKKp (encoded by Ikbkb), both of which are indispensable for canonical NF-<k>activation. b. We demonstrated that the immunosuppressive activity of Ly6C expressing v-FLAR cells isolated from Tg mice was significantly reduced after silencing iKKa or IKKp (Figure 11c). Taken together, our data indicate that c-FLAR activates a signaling pathway that controls an immunosuppressive program in monocytes through the activation of NF-κB.

Claims (12)

1. A population of cells for use in therapy, said population comprising myeloid cells or progenitor cells thereof that are designed to increase the cellular level of cellular FLICE [Fas-associated death domain-like IL-1p converting enzyme (FADD). )]- inhibitory protein (CFLAR), in which said population comprises myeloid cells or progenitor cells thereof that comprise a recombinant nucleic acid that encodes CFLAR, in which the therapy comprises treating a patient who needs immunosuppression or in which The therapy comprises treatment of a transplant recipient or graft versus host disease or an inflammatory disease or an autoimmune disease, wherein the inflammatory disease is preferably celiac disease, vasculitis, lupus, chronic obstructive pulmonary disease (COPD), irritable bowel disease, atherosclerosis, arthritis, ankylosing spondylitis, Crohn's disease, colitis, chronic active hepatitis, dermatitis or psoriasis and wherein the immune disease is preferably Addision's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, diabetes (Type I), dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barr syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, ulcerative colitis or diabetes mellitus type 2. The cell population for the use of claim 1, wherein the myeloid cells or their progenitor cells have been transfected using a viral vector comprising said nucleic acid encoding CFLAR. 3. The population of cells for use in claim 1, wherein said nucleic acid is RNA. 4. The cell population for use of any one of claims 1 to 3, wherein CFLAR is CFLAR and/or CFLARs. 5. The cell population for use of any one of claims 1 to 4, wherein the myeloid cells or progenitor cells thereof are isolated and/or purified cells, in particular isolated and/or purified by magnetic cell sorting, wherein the myeloid cells or progenitor cells thereof are preferably monocytes, more preferably CD14+ monocytes, or stem cells, or CD34+ bone marrow cells. 6. The cell population for use in any one of claims 1 to 5, wherein said bone marrow CD34+ cells have been isolated from bone marrow mononuclear cells (MNC). 7. A pharmaceutical composition for use in therapy, said pharmaceutical composition comprises a population of cells comprising myeloid cells or progenitor cells thereof that are designed to increase the cellular level of cellular FLICE inhibitor protein [Fas-associated death domain ( FADD) IL-1p converting enzyme-like] (CFLAR), wherein said population comprises myeloid cells or progenitor cells thereof comprising a recombinant nucleic acid encoding CFLAR wherein the therapy comprises treating a patient in need of immunosuppression or wherein the therapy comprises treatment of a transplant recipient or graft versus host disease or an inflammatory disease or an autoimmune disease, wherein the inflammatory disease is preferably celiac disease, vasculitis, lupus, obstructive pulmonary disease (COPD), irritable bowel disease, atherosclerosis, arthritis, ankylosing spondylitis, Crohn's disease, colitis, chronic active hepatitis, dermatitis or psoriasis and in which the autoimmune disease is preferably Addision's disease, alopecia areata, ankylosing spondylitis, hepatitis autoimmune, autoimmune mumps, Crohn's disease, diabetes (Type I), dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barr syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, ulcerative colitis or type I diabetes mellitus. 8. The pharmaceutical composition for use of claim 7 or the cell population for use of any of claims 1 to 6, wherein the cell population is autologous to the patient. 9. The pharmaceutical composition for use of claim 7 or 8, or the cell population for use of any of claims 1 to 6, comprising administering the pharmaceutical composition or the cell population. 10. A nucleic acid encoding CFLAR, or a pharmaceutical composition comprising said nucleic acid, for use in therapy, wherein the therapy comprises treating a patient in need of immunosuppression or wherein the therapy comprises treating a receptor for transplant or graft versus host disease or an inflammatory disease or an immune disease, wherein the inflammatory disease is preferably celiac disease, vasculitis, lupus, chronic obstructive pulmonary disease (COPD), irritable bowel disease, atherosclerosis, arthritis , ankylosing spondylitis, Crohn's disease, colitis, chronic active disease, hepatitis, dermatitis or psoriasis and in which the immune disease is preferably Addision's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, diabetes ( Type I), dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barr syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, ulcerative colitis or type I diabetes mellitus. 11. The CFLAR-encoding nucleic acid for use of claim 10, or the pharmaceutical composition for use of claim 10, wherein the therapy comprises administering the CFLAR-encoding nucleic acid or the pharmaceutical composition to a patient and wherein The nucleic acid transfects myeloid cells or progenitor cells thereof in the patient. 12. The CFLAR-encoding nucleic acid for use in claim 11, or the pharmaceutical composition for use in claim 11, wherein the nucleic acid is RNA or a viral vector, preferably a lentiviral vector.